Therapeutic polymeric nanoparticle compositions with high glass transition temperature or high molecular weight copolymers

ABSTRACT

The present disclosure relates in part to pharmaceutical compositions comprising polymeric nanoparticles having certain glass transition temperatures. Other aspects of the invention include methods of making such nanoparticles.

RELATED APPLICATIONS

This application claims priority to U.S. Ser. No. 61/286,559 filed Dec.15, 2009, U.S. Ser. No. 61/306,729 filed Feb. 22, 2010, U.S. Ser. No.61/405,778 filed Oct. 22, 2010, U.S. Ser. No. 61/286,831 filed Dec. 16,2009, and U.S. Ser. No. 61/286,897 filed Dec. 16, 2009, each of which ishereby incorporated by reference in its entirety.

BACKGROUND

Systems that deliver certain drugs to a patient (e.g., targeted to aparticular tissue or cell type or targeted to a specific diseased tissuebut not normal tissue), or that control release of drugs has long beenrecognized as beneficial. For example, therapeutics that include anactive drug and that are capable of locating in a particular tissue orcell type e.g., a specific diseased tissue, may reduce the amount of thedrug in tissues of the body that do not require treatment. This isparticularly important when treating a condition such as cancer where itis desirable that a cytotoxic dose of the drug is delivered to cancercells without killing the surrounding non-cancerous tissue. Further,such therapeutics may reduce the undesirable and sometimes lifethreatening side effects common in anticancer therapy. For example,nanoparticle therapeutics may, due the small size, evade recognitionwithin the body allowing for targeted and controlled delivery whilee.g., remaining stable for an effective amount of time.

Therapeutics that offer such therapy and/or controlled release and/ortargeted therapy also must be able to deliver an effective amount ofdrug. It can be a challenge to prepare nanoparticle systems that have anappropriate amount of drug associated with each nanoparticle, whilekeeping the size of the nanoparticles small enough to have advantageousdelivery properties. For example, while it is desirable to load ananoparticle with a high quantity of therapeutic agent, nanoparticlepreparations that use a drug load that is too high will result innanoparticles that are too large for practical therapeutic use. Further,it may be desirable for therapeutic nanoparticles to remain stable so asto e.g. substantially limit rapid or immediate release of thetherapeutic agent.

Accordingly, a need exists for new nanoparticle formulations and methodsof making such nanoparticles and compositions, that can delivertherapeutic levels of drugs to treat diseases such as cancer, while alsoreducing patient side effects.

SUMMARY

In one aspect, the disclosure provides a pharmaceutical aqueoussuspension comprising a plurality of nanoparticles, having a glasstransition temperature between about 37° C. and about 50° C., whereineach of the nanoparticles comprises a therapeutic agent and a blockcopolymer having at least one hydrophobic portion and at least onehydrophilic portion. The therapeutic agent may be a taxane agent, suchas docetaxel. The hydrophobic portion may be selected, for example, frompoly(D,L-lactic) acid and poly(lactic acid-co-glycolic acid). Thehydrophilic portion may be, for example, poly(ethylene)glycol. Thenanoparticles may further comprise poly(D,L-lactic) acid or poly(lactic)acid-co-poly(glycolic) acid.

In an embodiment, provided herein is a pharmaceutical aqueous suspensioncomprising a plurality of fast release biocompatible, therapeuticnanoparticles having a glass transition temperature between about 37° C.and about 38° C., wherein each of the nanoparticles comprises atherapeutic agent and a block copolymer having at least one hydrophobicportion and at least one hydrophilic portion. Such nanoparticles mayrelease about 70% to about 100% of the therapeutic agent at four hoursin an in vitro dissolution test.

In another embodiment, provided herein is a pharmaceutical aqueoussuspension comprising a plurality of moderate release biocompatible,therapeutic nanoparticles having a glass transition temperature betweenabout 39° C. and about 41° C., wherein each of the nanoparticlescomprises a therapeutic agent and a block copolymer having at least onehydrophobic portion and at least one hydrophilic portion. Suchtherapeutic nanoparticles may release between about 50% to about 70% ofthe therapeutic agent at four hours in an in vitro dissolution test.

In another embodiment, a pharmaceutical aqueous suspension is providedthat comprises a plurality of slow release biocompatible, therapeuticnanoparticles having a glass transition temperature between about 42° C.and about 50° C., wherein each of the nanoparticles comprises atherapeutic agent and a block copolymer having at least one hydrophobicportion and at least one hydrophilic portion. Such a therapeuticnanoparticle may release about 50% or less of the therapeutic agent atfour hours in an in vitro dissolution test.

In one embodiment, disclosed nanoparticles may comprise about 0.1 toabout 35, or 0.2 to about 20 weight percent of a therapeutic agent;about 10 to about 99 weight percent poly(D,L-lactic)acid-block-poly(ethylene)glycol copolymer orpoly(lactic)-co-poly(glycolic) acid-block-poly(ethylene)glycolcopolymer; and about 0 to about 75 weight percent, or about 0 to about50 weight percent, poly(D,L-lactic) acid or poly(lactic)acid-co-poly(glycolic) acid. In another embodiment, the poly(D,L-lactic)acid portion of the copolymer has a number average molecular weight ofabout 16 kDa, and the poly(ethylene)glycol portion of the copolymer hasa number average molecular weight of about 5 kDa. In an embodiment, thepoly(D,L-lactic) acid has a number average molecular weight of about 8.5kDa. In another embodiment, the poly(D,L-lactic) acid has a numbermolecular weight of about 75 kDa.

In one embodiment, the nanoparticle glass transition temperature, e.g.in a disclosed aqueous solution, may be about 37° C. to about 39° C., orabout 37° C. to about 38° C. In another embodiment, an aqueoussuspension of nanoparticles may have a glass transition temperature thatmay be about 39° C. to about 41° C., or may be about 42° C. to about 50°C. (e.g. about 41-45° C., e.g. for slow release particles). The glasstransition temperature may be measured by Heat Flux DifferentialScanning calorimetry or Power Compensation Differential Scanningcalorimetry.

In one embodiment, the disclosed nanoparticles release less than about50% of the therapeutic agent as determined in an in vitro dissolutiontest at a 4 hour time point (or optionally at a 1, 2, 8 or 24 hour timepoint). In another embodiment, the nanoparticles release between about50 to about 70% of the therapeutic agent as determined in an in vitrodissolution test at a 4 hour time point (or optionally at a 1, 2, 8 or24 hour time point). In another embodiment, the nanoparticles releasebetween about 70 to about 100% of the therapeutic agent as determined inan in vitro dissolution test at a 4 hour time point (or optionally at a1, 2, 8 or 24 hour time point).

In another aspect, the disclosure provides a method for determining thedrug release rate of a therapeutic polymeric nanoparticle composition,comprising: a) providing at least one first plurality of polymericnanoparticles comprising a first therapeutic agent, a first blockcopolymer having at least one hydrophobic portion and at least onehydrophilic portion, and optionally poly(D,L-lactic) acid orpoly(lactic) acid-co-poly(glycolic) acid; b) determining thenanoparticle glass transition temperature for the at least one firstplurality of polymeric nanoparticles; c) determining the drug releaserate from the at least one first plurality of polymeric nanoparticles;and d) determining the correlation between the nanoparticle glasstransition temperature and the drug release rate for the at least onefirst plurality of polymeric nanoparticles.

A method for screening nanoparticle suspensions is also provided,comprising: i) providing a suspension comprising a first plurality ofpolymeric nanoparticles, wherein the nanoparticles each comprise atherapeutic agent, a block copolymer having at least one hydrophobicportion and at least one hydrophilic portion, and a homopolymer selectedfrom poly(D,L-lactic) acid or poly(lactic) acid-co-poly(glycolic) acid;ii) determining the glass transition temperature for the suspension;iii) increasing or decreasing the amount of the homopolymer in the firstplurality of polymeric nanoparticles; and iv) repeating steps i)-iii)until a suspension with a desired glass transition temperature isachieved.

A method for screening nanoparticle suspensions to identify a suspensionhaving a specific release rate is provided, comprising: a) separatelypreparing a plurality of suspensions having nanoparticles comprising atherapeutic agent, a block copolymer having at least one hydrophobicportion and at least one hydrophilic portion, and optionally ahomopolymer selected from poly(D,L-lactic) acid or poly(lactic)acid-co-poly(glycolic) acid; wherein each suspension is in a separatecompartment, each suspension comprises a pre-determined molecular weightof the block copolymer and if present, a pre-determined molecular weightof the homopolymer; b) determining the glass transition temperature ofeach of the suspensions; c) identifying the suspension having apre-determined glass transition temperature.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a flow chart for an emulsion process for forming disclosednanoparticles.

FIGS. 2A and 2B are flow diagrams for a disclosed emulsion process. FIG.2A shows particle formation and hardening (upstream processing). FIG. 2Bshows particle work up and purification (downstream processing)

FIG. 3 is a DSC curve of poly(D,L-lactide)-block-poly(ethylene glycol)(PLA-PEG, Mn PLA block=16 kDa; Mn PEG block=5 kDa) when recovered from amelt polymerization and having been cooled at an unknown cooling rate.

FIG. 4A is a DSC curve of poly(D,L-lactide)-block-poly(ethylene glycol)(PLA-PEG, Mn PLA block=16 kDa; Mn PEG block=5 kDa) when recovered from aprecipitation of polymer solution (100 mg/mL in dichloromethane) into abinary non-solvent mixture (diethyl ether/hexane=70/30 (v/v); FIG. 4Bare DSC curves showing glass transitions observed in PLA-PEG blockcopolymers of increasing molecular weights. PLA block number averagemolecular weight, Mn=10 KDa (lower curve), 15 KDa, 30 KDa and 50 KDa;FIG. 4C shows the dependence of Tg on the molecular weight (M_(n)) ofPLA in PLA-PEG block copolymers.

FIG. 5 is a DSC curve of poly(D,L-lactide) (PLA, Mn=6 kDa) whenrecovered from a precipitation process.

FIG. 6A depicts a DSC curve of poly(D,L-lactide) (PLA, Mn=75 kDa) whenrecovered from a precipitation process; FIG. 6B depicts modulated DSCcurves showing the glass transition temperatures in homopolymerpoly(D,L-lactide) of number average molecular weights (M_(n)) 1.7 KDa(lower), 4.3 KDa, 6 KDa, 10 KDa, 22 KDa, and 120 KDa; FIG. 6C shows thedependence of Tg on the number average molecular weight (M_(n)) of PLAhomopolymers

FIG. 7 is an illustration of the five points used to define theendothermic transitions observed in the DSC analysis of nanoparticles.

FIG. 8 is a DSC curve showing the endothermic glass transition observedin nanoparticles composed of a mixture of PLA-PEG (Mn PLA block=16 kDa;Mn PEG block=5 kDa) and low molecular weight PLA homopolymer (Mn=6.5kDa).

FIG. 9 is a DSC curve showing the endothermic glass transition observedin nanoparticles composed of PLA-PEG (Mn PLA block=16 kDa; Mn PEGblock=5 kDa) as the only polymeric component of the particle.

FIG. 10 is a DSC curve showing the endothermic glass transition observedin nanoparticles composed of a mixture of PLA-PEG (Mn PLA block=16 kDa;M_(n) PEG block=5 kDa) and high molecular weight PLA homopolymer(M_(n)=75 kDa).

FIGS. 11A and 11B show DSC curves showing the endothermic glasstransition observed in nanoparticles composed of a mixture of PLA-PEG(16 kDa-5 kDa) and high molecular weight PLA homopolymer (M_(n)—75 kDa).

FIG. 12 is a comparison of docetaxel (DTXL) release rates fromnanoparticles based on different polymeric components as detailed in theplot legend.

FIG. 13 is a graph showing the effect of temperature on drug releaserates over 24 hours from nanoparticle systems that exhibit differentglass transition temperatures.

FIG. 14 is a graph showing an expansion of the 1-4 hour time period ofthe study given in FIG. 13.

FIG. 15 depicts in vitro release of docetaxel of various nanoparticlesdisclosed herein.

FIG. 16 depicts in vitro release of bortezomib of various nanoparticlesdisclosed herein.

FIG. 17 depicts in vitro release of vinorelbine of various nanoparticlesdisclosed herein.

FIG. 18 depicts in vitro release of vincristine of various nanoparticlesdisclosed herein.

FIG. 19 depicts in vitro release of bendamustine HCl of variousnanoparticles disclosed herein.

FIG. 20 depicts in-vitro release of epothilone B of variousnanoparticles disclosed herein.

FIG. 21 depicts in-vitro release of budesonide of various nanoparticlesdisclosed herein.

FIG. 22 depicts in-vitro release of budesonide of various nanoparticlesdisclosed herein.

FIG. 23 depicts pharmacokinetics of budesonide and budesonidenanoparticles following a singe intravenous dose (0.5 mg/kg).

FIG. 24 indicates disease scores in rat intestines in a model of IBDafter treatment with budesonide, budesonide PTNP and dexamethasone.

FIG. 25 indicates rat intestinal weights in a Model of IBD afterTreatment with budesonide, budesonide PTNP and dexamethasone.

FIG. 26 depicts in vitro release of budesonide in various nanoparticles.

DETAILED DESCRIPTION

At least in part, this disclosure relates to fast releasingbiocompatible, therapeutic nanoparticles having a glass transitiontemperature between about 37° C. and about 38° C., and/or pharmaceuticalaqueous suspensions that include a plurality of moderate releasingbiocompatible, therapeutic nanoparticles having a glass transitiontemperature between about 39° C. and about 41° C., and/or slow releasingpharmaceutical aqueous suspensions that include a plurality ofbiocompatible, therapeutic nanoparticles having a glass transitiontemperature between about 42° C. and about 50° C. (or about 42 to about45° C.). Disclosed nanoparticles include a therapeutic agent and mayinclude a block copolymer having at least one hydrophobic portion and atleast one hydrophilic portion.

For example, provided herein is a pharmaceutical aqueous suspensioncomprising a plurality of fast release biocompatible, therapeuticnanoparticles having a glass transition temperature between about 37° C.and about 38° C., wherein each of the nanoparticles comprises atherapeutic agent and a block copolymer having at least one hydrophobicportion and at least one hydrophilic portion wherein the nanoparticlesrelease about 70% to about 100% of the therapeutic agent at four hoursin an in vitro dissolution test. Also provided herein is apharmaceutical aqueous suspension comprising a plurality of moderaterelease biocompatible, therapeutic nanoparticles having a glasstransition temperature between about 39° C. and about 41° C., whereineach of the nanoparticles comprises a therapeutic agent and a blockcopolymer having at least one hydrophobic portion and at least onehydrophilic portion, and wherein the nanoparticles release between about50% to about 70% of the therapeutic agent at four hours in an in vitrodissolution test. In another embodiment, a pharmaceutical aqueoussuspension is provided that comprises a plurality of slow releasebiocompatible, therapeutic nanoparticles having a glass transitiontemperature between about 42° C. and about 50° C., wherein each of thenanoparticles comprises a therapeutic agent and a block copolymer havingat least one hydrophobic portion and at least one hydrophilic portion,wherein the nanoparticles release about 50% or less of the therapeuticagent at four hours in an in vitro dissolution test. Such dissolutiontests are well known in the art. One such representative test isexemplified below in Example 7. For example, a dissolution test mayinclude placing the suspension 2.5% wt hydroxypropyl cyclodextrinphosphate buffer saline, (e.g. 0.01M phosphate buffered saline) for 1,4, 8, 12 days or more.

In general, disclosed compositions may include nanoparticles thatinclude an active agent.

Disclosed nanoparticles may include about 0.1 to about 40 weightpercent, 0.2 to about 35 weight percent, about 3 to about 40 weightpercent, about 5 to about 30 weight percent, 10 to about 30 weightpercent, 15 to 25 weight percent, or even about 4 to about 25 weightpercent of an active agent, such as antineoplastic agent, e.g., a taxaneagent (for example, docetaxel).

Nanoparticles disclosed herein include one, two, three or morebiocompatible and/or biodegradable polymers, such as described herein.For example, a contemplated nanoparticle may include about 10 to about99 weight percent of one or more block co-polymers that include abiodegradable polymer and polyethylene glycol, and about 0 to about 50weight percent, or about 0 to about 75 weight percent of a biodegradablehomopolymer, e.g. PLA.

Exemplary therapeutic nanoparticles may include about 40 to about 99, orabout 50 to about 90 weight percent poly(lactic)acid-poly(ethylene)glycol copolymer or about 40 to about 80 weightpercent poly(lactic) acid-poly(ethylene)glycol copolymer. Suchpoly(lactic) acid-block-poly(ethylene)glycol copolymer may includepoly(lactic acid) having a number average molecular weight of about 15to 20 kDa (or for example about 15 to about 100 kDa, e.g., about 15 toabout 80 kDa), and poly(ethylene)glycol having a number averagemolecular weight of about 2 to about 10 kDa, for example, about 4 toabout 6 kDa. For example, a disclosed therapeutic nanoparticle mayinclude about 70 to about 90 weight percent PLA-PEG and about 15 toabout 25 weight percent active agent, or about 30 to about 50 weightpercent PLA-PEG, about 30 to about 50 weight percent (or about 30 toabout 75 weight percent) PLA or PLGA, and about 15 to about 25 weightpercent active agent. Such PLA ((poly)lactic acid) may have a numberaverage molecular weight of about 5 to about 10 kDa. Such PLGA (polylactic-co-glycolic acid) may have a number average molecular weight ofabout 8 to about 12 kDa.

In other embodiments, nanoparticles disclosed herein include one or morebiocompatible and/or biodegradable polymers, for example, a highmolecular weight diblock poly(lactic) acid-poly(ethylene)glycolcopolymer or a high molecular weight diblockpoly(lactic)-co-poly(glycolic) acid-poly(ethylene)glycol copolymer. Forexample, a diblock poly(lactic) acid-poly(ethylene)glycol copolymercomprises poly(lactic) acid may have a number average molecule weight ofabout 30 kDa to about 90 kDa, or about 40 kDa to about 90 kDa. Inanother embodiment, a diblock poly(lactic)-co-poly(glycolic)acid-poly(ethylene)glycol copolymer comprisespoly(lactic)-co-poly(glycolic) acid having a number average moleculeweight of about 30 kDa to about 90 kDa, or about 40 kDa to about 90 kDa.For example, a contemplated nanoparticle may include about 0.1 to about40 weight percent of a therapeutic agent and about 10 to about 90 weightpercent a diblock poly(lactic) acid-poly(ethylene)glycol copolymer,wherein the diblock poly(lactic) acid-poly(ethylene)glycol copolymercomprises poly(lactic) acid having a number average molecule weight ofabout 30 kDa to about 90 kDa, or about 40 kDa to about 90 kDa. In oneembodiment, the poly(lactic) acid has a number average molecule weightof about 30 kDa. In another embodiment, the poly(lactic) acid has anumber average molecule weight of about 50 kDa to about 80 kDa, or about70 kDa to about 85 kDa. In yet another embodiment, the poly(lactic) acidhas a number average molecule weight of about 50 kDa. In someembodiments, the diblock poly(lactic) acid-poly(ethylene)glycolcopolymer or the diblock poly(lactic)-co-poly(glycolic)acid-poly(ethylene)glycol copolymer comprises poly(ethylene)glycolhaving a molecular weight of about 4 kDa to about 6 kDa, or about 4 kDato about 12 kDa. For example, the poly(ethylene)glycol may have a numberaverage molecule weight of about 5 kDa or 10 kDa.

Disclosed nanoparticles may optionally include about 1 to about 50weight percent or about 1 to about 70 weight percent poly(lactic) acidor poly(lactic) acid-co-poly (glycolic) acid (which does not includePEG, e.g a homopolymer of PLA), or may optionally include about 1 toabout 75 weight percent, or about 10 to about 50 weight percent or about30 to about 50 weight percent poly(lactic) acid or poly(lactic)acid-co-poly(glycolic) acid. In an embodiment, disclosed nanoparticlesmay include two polymers, e.g. PLA-PEG and PLA, in a weight ratio ofabout 40:60 to about 60:40, or about 30:50 to about 50:30, e.g, about50:50.

Such substantially homopolymeric poly(lactic) orpoly(lactic)-co-poly(glycolic) acid may have a weight average molecularweight of about 4.5 to about 130 kDa, for example, about 20 to about 30kDa, or about 100 to about 130 kDa. Such homopolymeric PLA may have anumber average molecule weight of about 4.5 to about 90 kDa, or about4.5 to about 12 kDa, about 5.5 to about 7 kDa (e.g. about 6.5 kDa),about 15 to about 30 kDa, or about 60 to about 90 kDa. Exemplaryhomopolymeric PLA may have a number average molecular weight of about 70or 80 kDa or a weight average molecular weight of about 124 kD. As isknown in the art, molecular weight of polymers can be related to aninherent viscosity. In some embodiments, homopolymer PLA may have aninherent viscosity of about 0.2 to about 0.4, e.g. about 0.4; in otherembodiments, PLA may have an inherent viscosity of about 0.6 to about0.8. Exemplary PLGA may have a number average molecular weight of about8 to about 12 kDa.

For example, provided herein is abiocompatible, therapeutic polymericnanoparticle comprising about 0.1 to about 40 weight percent of atherapeutic agent; and about 10 to about 90, or about 10 to about 99, orabout 70 to about 99 weight percent biocompatible polymer, wherein thebiocompatible polymer is selected from the group consisting of a) adiblock poly(lactic) acid-poly(ethylene)glycol copolymer, wherein thediblock poly(lactic) acid-poly(ethylene)glycol copolymer comprisespoly(lactic) acid having a number average molecule weight of about 30kDa to about 90 kDa; and b) a diblock poly(lactic)-co-poly(glycolic)acid-poly(ethylene)glycol copolymer, wherein the diblockpoly(lactic)-co-poly(glycolic) acid-poly(ethylene)glycol copolymercomprises poly(lactic)-co-poly(glycolic) acid having a number averagemolecule weight of about 30 kDa to about 90 kDa. The diblockpoly(lactic) acid-poly(ethylene)glycol copolymer or the diblockpoly(lactic)-co-poly(glycolic) acid-poly(ethylene)glycol copolymer mayinclude poly(ethylene)glycol having a molecular weight of about 4 kDa toabout 12 kDa, for example, a diblock poly(lactic)acid-poly(ethylene)glycol copolymer may include, poly(lactic) acidhaving a number average molecular weight of about 30 kDa andpoly(ethylene)glycol having a number average molecular weight of about 5kDa, or may include poly(lactic) acid having a number average molecularweight of about 50 kDa to about 80 kDa and poly(ethylene)glycol having anumber average molecular weight of about 5 kDa or 10 kDa, e.g.,poly(lactic) acid having a number average molecular weight of about 50kDa and poly(ethylene)glycol having a number average molecular weight ofabout 5 kDa.

In one embodiment, disclosed therapeutic nanoparticles may include atargeting ligand, e.g., a low-molecular weight PSMA ligand effective forthe treatment of a disease or disorder, such as prostate cancer, in asubject in need thereof. In certain embodiments, the low-molecularweight ligand is conjugated to a polymer, and the nanoparticle comprisesa certain ratio of ligand-conjugated polymer (e.g., PLA-PEG-Ligand) tonon-functionalized polymer (e.g., PLA-PEG or PLGA-PEG). The nanoparticlecan have an optimized ratio of these two polymers such that an effectiveamount of ligand is associated with the nanoparticle for treatment of adisease or disorder, such as cancer.

In some embodiments, disclosed nanoparticles may further comprise about0.2 to about 10 weight percent PLA-PEG functionalized with a targetingligand such as disclosed herein, and/or may include about 0.2 to about10 weight percent poly(lactic) acid-co poly (glycolic) acidblock-PEG-functionalized with a targeting ligand. Such a targetingligand may be, in some embodiments, covalently bound to the PEG, forexample, bound to the PEG via an alkylene linker, e.g.,PLA-PEG-alkylene-ligand. For example, a disclosed nanoparticle mayinclude about 0.2 to about 10 mole percent PLA-PEG-ligand orpoly(lactic) acid-co poly (glycolic) acid-PEG-ligand.)

In some embodiments, disclosed therapeutic particles and/or compositionsinclude targeting or imaging agents such as dyes, for example Evans bluedye. Such dyes may be bound to or associated with a therapeuticparticle, or disclosed compositions may include such dyes. For example,Evans blue dye may be used, which may bind or associate with albumin,e.g. plasma albumin.

Disclosed nanoparticles may have a substantially spherical (i.e., theparticles generally appear to be spherical), or non-sphericalconfiguration. For instance, the particles, upon swelling or shrinkage,may adopt a non-spherical configuration.

Disclosed nanoparticles may have a characteristic dimension of less thanabout 1 micrometer, where the characteristic dimension of a particle isthe diameter of a perfect sphere having the same volume as the particle.For example, the particle can have a characteristic dimension of theparticle can be less than about 300 nm, less than about 200 nm, lessthan about 150 nm, less than about 100 nm, less than about 50 nm, lessthan about 30 nm, less than about 10 nm, less than about 3 nm, or lessthan about 1 nm in some cases. In particular embodiments, disclosednanoparticles may have a diameter of about 70 nm to about 250 nm, orabout 70 nm to about 180 nm, about 80 nm to about 170 nm, about 80 nm toabout 130 nm.

In one set of embodiments, the particles can have an interior and asurface, where the surface has a composition different from theinterior, i.e., there may be at least one compound present in theinterior but not present on the surface (or vice versa), and/or at leastone compound is present in the interior and on the surface at differingconcentrations. For example, in one embodiment, a compound, such as atargeting moiety (i.e., a low-molecular weight ligand) of a polymericconjugate of the present invention, may be present in both the interiorand the surface of the particle, but at a higher concentration on thesurface than in the interior of the particle, although in some cases,the concentration in the interior of the particle may be essentiallynonzero, i.e., there is a detectable amount of the compound present inthe interior of the particle.

In some cases, the interior of the particle is more hydrophobic than thesurface of the particle. For instance, the interior of the particle maybe relatively hydrophobic with respect to the surface of the particle,and a drug or other payload may be hydrophobic, and readily associateswith the relatively hydrophobic center of the particle. The drug orother payload can thus be contained within the interior of the particle,which can shelter it from the external environment surrounding theparticle (or vice versa). For instance, a drug or other payloadcontained within a particle administered to a subject will be protectedfrom a subject's body, and the body may also be substantially isolatedfrom the drug for at least a period of time.

Disclosed nanoparticles may be stable, for example in a solution thatmay contain a saccharide, for at least about 24 hours, about 2 days, 3days, about 4 days or at least about 5 days at room temperature, or at25° C.

Nanoparticles disclosed herein may have controlled release properties,e.g., may be capable of delivering an amount of active agent to apatient, e.g., to specific site in a patient, over an extended period oftime, e.g. over 1 day, 1 week, or more.

DEFINITIONS

“Treating” includes any effect, e.g., lessening, reducing, modulating,or eliminating, that results in the improvement of the condition,disease, disorder and the like.

“Pharmaceutically or pharmacologically acceptable” describes molecularentities and compositions that do not produce an adverse, allergic orother untoward reaction when administered to an animal, or a human, asappropriate. For human administration, preparations should meetsterility, pyrogenicity, general safety and purity standards as requiredby FDA Office of Biologics standards.

The term “pharmaceutically acceptable carrier” or “pharmaceuticallyacceptable excipient” as used herein refers to any and all solvents,dispersion media, coatings, isotonic and absorption delaying agents, andthe like, that are compatible with pharmaceutical administration. Theuse of such media and agents for pharmaceutically active substances iswell known in the art. The compositions may also contain other activecompounds providing supplemental, additional, or enhanced therapeuticfunctions.

“Individual,” “patient,” or “subject” are used interchangeably andinclude any animal, including mammals, such as mice, rats, otherrodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates,and most preferably humans. The compounds and compositions of theinvention can be administered to a mammal, such as a human, but can alsobe other mammals such as an animal in need of veterinary treatment,e.g., domestic animals (e.g., dogs, cats, and the like), farm animals(e.g., cows, sheep, pigs, horses, and the like) and laboratory animals(e.g., rats, mice, guinea pigs, and the like). “Modulation” includesantagonism (e.g., inhibition), agonism, partial antagonism and/orpartial agonism.

In the present specification, the term “therapeutically effectiveamount” means the amount of the subject compound or composition thatwill elicit the biological or medical response of a tissue, system,animal or human that is being sought by the researcher, veterinarian,medical doctor or other clinician. The compounds and compositions of theinvention are administered in therapeutically effective amounts to treata disease. Alternatively, a therapeutically effective amount of acompound is the quantity required to achieve a desired therapeuticand/or prophylactic effect.

The term “pharmaceutically acceptable salt(s)” as used herein refers tosalts of acidic or basic groups that may be present in compounds used inthe present compositions. Compounds included in the present compositionsthat are basic in nature are capable of forming a wide variety of saltswith various inorganic and organic acids. The acids that may be used toprepare pharmaceutically acceptable acid addition salts of such basiccompounds are those that form non-toxic acid addition salts, i.e., saltscontaining pharmacologically acceptable anions, including but notlimited to malate, oxalate, chloride, bromide, iodide, nitrate, sulfate,bisulfate, phosphate, acid phosphate, isonicotinate, acetate, lactate,salicylate, citrate, tartrate, oleate, tannate, pantothenate,bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate,gluconate, glucaronate, saccharate, formate, benzoate, glutamate,methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonateand pamoate (i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts.Compounds included in the present compositions that include an aminomoiety may form pharmaceutically acceptable salts with various aminoacids, in addition to the acids mentioned above. Compounds included inthe present compositions that are acidic in nature are capable offorming base salts with various pharmacologically acceptable cations.Examples of such salts include alkali metal or alkaline earth metalsalts, such as calcium, magnesium, sodium, lithium, zinc, potassium, andiron salts

Polymers

In some embodiments, the nanoparticles disclosed herein include a matrixof polymers and a therapeutic agent.

Contemplated herein are nanoparticles comprising polymers, for example,copolymers. Various molecular weights of polymers are contemplatedherein, for example, the weight of a polymer may influence particledegradation rate, solubility, water uptake, and drug release kinetics.The molecular weight of the polymer can be adjusted such that theparticle biodegrades in the subject being treated within a reasonableperiod of time (ranging from a few hours to 1-2 weeks, 3-4 weeks, 5-6weeks, 7-8 weeks, etc.) For example, a disclosed particle may comprise acopolymer of PLA and PEG or PLGA and PEG, wherein the PLA or PLGAportion may have a number average molecule weight of about 30 kDa toabout 90 kDa or about 40 kDa to about 90 kDa, and the PEG portion mayhave a molecular weight of about 4 kDa to about 6 kDa. In an exemplaryembodiment, the PLA or the PLGA portion may have a number averagemolecule weight of 30 kDa, 50 kDa, 65 kDa, or 80 kDa. The PEG potion mayhave a molecular weight of about 5 kDa, about 6, 7, 8, or 9 kDa, orabout 10 kDa.

Disclosed nanoparticles may include one or more polymers, e.g. a firstpolymer that may be a co-polymer, e.g. a diblock co-polymer, andoptionally a polymer that may be for example a homopolymer. In someembodiments, disclosed nanoparticles include a matrix of polymers.Disclosed therapeutic nanoparticles may include a therapeutic agent thatcan be associated with the surface of, encapsulated within, surroundedby, and/or dispersed throughout a polymeric matrix.

Disclosed particles can include copolymers, which, in some embodiments,describes two or more polymers (such as those described herein) thathave been associated with each other, usually by covalent bonding of thetwo or more polymers together. Thus, a copolymer may comprise a firstpolymer and a second polymer, which have been conjugated together toform a block copolymer where the first polymer can be a first block ofthe block copolymer and the second polymer can be a second block of theblock copolymer. Of course, those of ordinary skill in the art willunderstand that a block copolymer may, in some cases, contain multipleblocks of polymer, and that a “block copolymer,” as used herein, is notlimited to only block copolymers having only a single first block and asingle second block. For instance, a block copolymer may comprise afirst block comprising a first polymer, a second block comprising asecond polymer, and a third block comprising a third polymer or thefirst polymer, etc. In some cases, block copolymers can contain anynumber of first blocks of a first polymer and second blocks of a secondpolymer (and in certain cases, third blocks, fourth blocks, etc.). Inaddition, it should be noted that block copolymers can also be formed,in some instances, from other block copolymers. For example, a firstblock copolymer may be conjugated to another polymer (which may be ahomopolymer, a biopolymer, another block copolymer, etc.), to form a newblock copolymer containing multiple types of blocks, and/or to othermoieties (e.g., to non-polymeric moieties).

In some embodiments, a contemplated copolymer (e.g., block copolymer)can be amphiphilic, i.e., having a hydrophilic portion and a hydrophobicportion, or a relatively hydrophilic portion and a relativelyhydrophobic portion. A hydrophilic polymer can be one generally thatattracts water and a hydrophobic polymer can be one that generallyrepels water. A hydrophilic or a hydrophobic polymer can be identified,for example, by preparing a sample of the polymer and measuring itscontact angle with water (typically, the polymer will have a contactangle of less than 60°, while a hydrophobic polymer will have a contactangle of greater than about 60°). In some cases, the hydrophilicity oftwo or more polymers may be measured relative to each other, i.e., afirst polymer may be more hydrophilic than a second polymer. Forinstance, the first polymer may have a smaller contact angle than thesecond polymer.

In one set of embodiments, a high molecular weight polymer (e.g.,copolymer, e.g., block copolymer) contemplated herein includes abiocompatible polymer, i.e., the polymer that does not typically inducean adverse response when inserted or injected into a living subject, forexample, without significant inflammation and/or acute rejection of thepolymer by the immune system, for instance, via a T-cell response.Accordingly, the therapeutic particles contemplated herein can benon-immunogenic. The term non-immunogenic as used herein refers toendogenous growth factor in its native state which normally elicits no,or only minimal levels of, circulating antibodies, T-cells, or reactiveimmune cells, and which normally does not elicit in the individual animmune response against itself.

Biocompatibility typically refers to the acute rejection of material byat least a portion of the immune system, i.e., a nonbiocompatiblematerial implanted into a subject provokes an immune response in thesubject that can be severe enough such that the rejection of thematerial by the immune system cannot be adequately controlled, and oftenis of a degree such that the material must be removed from the subject.One simple test to determine biocompatibility can be to expose a polymerto cells in vitro; biocompatible polymers are polymers that typicallywill not result in significant cell death at moderate concentrations,e.g., at concentrations of 50 micrograms/10⁶ cells. For instance, abiocompatible polymer may cause less than about 20% cell death whenexposed to cells such as fibroblasts or epithelial cells, even ifphagocytosed or otherwise uptaken by such cells. Non-limiting examplesof biocompatible polymers that may be useful in various embodiments ofthe present invention include polydioxanone (PDO), polyhydroxyalkanoate,polyhydroxybutyrate, poly(glycerol sebacate), polyglycolide,polylactide, PLGA, PLA, polycaprolactone, or copolymers or derivativesincluding these and/or other polymers.

In certain embodiments, contemplated biocompatible polymers may bebiodegradable, i.e., the polymer is able to degrade, chemically and/orbiologically, within a physiological environment, such as within thebody. As used herein, “biodegradable” polymers are those that, whenintroduced into cells, are broken down by the cellular machinery(biologically degradable) and/or by a chemical process, such ashydrolysis, (chemically degradable) into components that the cells caneither reuse or dispose of without significant toxic effect on thecells. In one embodiment, the biodegradable polymer and theirdegradation byproducts can be biocompatible.

For instance, a contemplated polymer may be one that hydrolyzesspontaneously upon exposure to water (e.g., within a subject), thepolymer may degrade upon exposure to heat (e.g., at temperatures ofabout 37° C.). Degradation of a polymer may occur at varying rates,depending on the polymer or copolymer used. For example, the half-lifeof the polymer (the time at which 50% of the polymer can be degradedinto monomers and/or other nonpolymeric moieties) may be on the order ofdays, weeks, months, or years, depending on the polymer. The polymersmay be biologically degraded, e.g., by enzymatic activity or cellularmachinery, in some cases, for example, through exposure to a lysozyme(e.g., having relatively low pH). In some cases, the polymers may bebroken down into monomers and/or other nonpolymeric moieties that cellscan either reuse or dispose of without significant toxic effect on thecells (for example, polylactide may be hydrolyzed to form lactic acid,polyglycolide may be hydrolyzed to form glycolic acid, etc.).

In some embodiments, polymers may be polyesters, including copolymerscomprising lactic acid and glycolic acid units, such aspoly(lactic)-co-poly(glycolic) acid, poly(lactic acid-co-glycolic acid),and poly(lactide-co-glycolide), collectively referred to herein as“PLGA”; and homopolymers comprising glycolic acid units, referred toherein as “PGA,” and lactic acid units, such as poly-L-lactic acid,poly-D-lactic acid, poly-D,L-lactic acid, poly-L-lactide,poly-D-lactide, and poly-D,L-lactide, collectively referred to herein as“PLA.” In some embodiments, exemplary polyesters include, for example,polyhydroxyacids or polyanhydrides.

In other embodiments, contemplated polyesters for use in disclosednanoparticles may be diblock copolymers, e.g., PEGylated polymers andcopolymers (containing poly(ethylene glycol) repeat units) such as oflactide and glycolide (e.g., PEGylated PLA, PEGylated PGA, PEGylatedPLGA), PEGylated poly(caprolactone), and derivatives thereof. Forexample, a “PEGylated” polymer may assist in the control of inflammationand/or immunogenicity (i.e., the ability to provoke an immune response)and/or lower the rate of clearance from the circulatory system via thereticuloendothelial system (RES), due to the presence of thepoly(ethylene glycol) groups.

PEGylation may also be used, in some cases, to decrease chargeinteraction between a polymer and a biological moiety, e.g., by creatinga hydrophilic layer on the surface of the polymer, which may shield thepolymer from interacting with the biological moiety. In some cases, theaddition of poly(ethylene glycol) repeat units may increase plasmahalf-life of the polymer (e.g., copolymer, e.g., block copolymer), forinstance, by decreasing the uptake of the polymer by the phagocyticsystem while decreasing transfection/uptake efficiency by cells. Thoseof ordinary skill in the art will know of methods and techniques forPEGylating a polymer, for example, by using EDC(1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) andNHS(N-hydroxysuccinimide) to react a polymer to a PEG group terminatingin an amine, by ring opening polymerization techniques (ROMP), or thelike.

Other contemplated polymers that may form part of a disclosednanoparticle may include poly(ortho ester) PEGylated poly(ortho ester),polylysine, PEGylated polylysine, poly(ethylene imine), PEGylatedpoly(ethylene imine), poly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester), poly[α-(4-aminobutyl)-L-glycolic acid],and derivatives thereof. In other embodiments, polymers can bedegradable polyesters bearing cationic side chains. Examples of thesepolyesters include poly(L-lactide-co-L-lysine), poly(serine ester),poly(4-hydroxy-L-proline ester).

In other embodiments, polymers may be one or more acrylic polymers. Incertain embodiments, acrylic polymers include, for example, acrylic acidand methacrylic acid copolymers, methyl methacrylate copolymers,ethoxyethyl methacrylates, cyanoethyl methacrylate, amino alkylmethacrylate copolymer, poly(acrylic acid), poly(methacrylic acid),methacrylic acid alkylamide copolymer, poly(methyl methacrylate),poly(methacrylic acid polyacrylamide, amino alkyl methacrylatecopolymer, glycidyl methacrylate copolymers, polycyanoacrylates, andcombinations comprising one or more of the foregoing polymers. Theacrylic polymer may comprise fully-polymerized copolymers of acrylic andmethacrylic acid esters with a low content of quaternary ammoniumgroups.

PLGA contemplated for use as described herein can be characterized by alactic acid:glycolic acid ratio of e.g., approximately 85:15,approximately 75:25, approximately 60:40, approximately 50:50,approximately 40:60, approximately 25:75, or approximately 15:85. Insome embodiments, the ratio of lactic acid to glycolic acid monomers inthe polymer of the particle (e.g., a PLGA block copolymer or PLGA-PEGblock copolymer), may be selected to optimize for various parameterssuch as water uptake, therapeutic agent release and/or polymerdegradation kinetics can be optimized. In other embodiments, the endgroup of a PLA polymer chain may be a carboxylic acid group, an aminegroup, or a capped end group with e.g., a long chain alkyl group orcholesterol.

Targeting Moieties

Provided herein are nanoparticles that may include an optional targetingmoiety, i.e., a moiety able to bind to or otherwise associate with abiological entity, for example, a membrane component, a cell surfacereceptor, prostate specific membrane antigen, or the like. A targetingmoiety present on the surface of the particle may allow the particle tobecome localized at a particular targeting site, for instance, a tumor,a disease site, a tissue, an organ, a type of cell, etc. As such, thenanoparticle may then be “target specific.” The drug or other payloadmay then, in some cases, be released from the particle and allowed tointeract locally with the particular targeting site.

For example, a targeting portion may cause the particles to becomelocalized to a tumor (e.g., a solid tumor) a disease site, a tissue, anorgan, a type of cell, etc. within the body of a subject, depending onthe targeting moiety used. For example, a low-molecular weight PSMAligand may become localized to a solid tumor, e.g., breast or prostatetumors or cancer cells. The subject may be a human or non-human animal.Examples of subjects include, but are not limited to, a mammal such as adog, a cat, a horse, a donkey, a rabbit, a cow, a pig, a sheep, a goat,a rat, a mouse, a guinea pig, a hamster, a primate, a human or the like.

For example, a contemplated low-molecular weight PSMA ligand that, e.g.,may be conjugated to a disclosed copolymer (and thus form, in someembodiments, part of a disclosed nanoparticle) may be represented by:

and enantiomers, stereoisomers, rotamers, tautomers, diastereomers, orracemates thereof.

Therapeutic Agents

According to the present invention, any agents including, for example,therapeutic agents (e.g. anti-cancer agents), diagnostic agents (e.g.contrast agents; radionuclides; and fluorescent, luminescent, andmagnetic moieties), prophylactic agents (e.g. vaccines), and/ornutraceutical agents (e.g. vitamins, minerals, etc.) may be delivered bythe disclosed nanoparticles. Exemplary agents to be delivered inaccordance with the present invention include, but are not limited to,small molecules (e.g. cytotoxic agents), nucleic acids (e.g., siRNA,RNAi, and mircoRNA agents), proteins (e.g. antibodies), peptides,lipids, carbohydrates, hormones, metals, radioactive elements andcompounds, drugs, vaccines, immunological agents, etc., and/orcombinations thereof. In some embodiments, the agent to be delivered isan agent useful in the treatment of cancer (e.g., breast, lung, orprostate cancer).

The active agent or drug may be a therapeutic agent such as mTorinhibitors (e.g., sirolimus, temsirolimus, or everolimus), vincaalkaloids (e.g. vinorelbine or vincristine), a diterpene derivative, ataxane (e.g. paclitaxel or its derivatives such as DHA-paclitaxel orPG-paxlitaxelor, or docetaxel), a boronate ester or peptide boronic acidcompound (e.g. bortezomib), a cardiovascular agent (e.g. a diuretic, avasodilator, angiotensin converting enzyme, a beta blocker, analdosterone antagonist, or a blood thinner), a corticosteroid (e.g.budensonide, fluocinonide, triamcinolone, mometasone, amcinonide,halcinonide, ciclesonide, beclomethansone), an antimetabolite orantifolate agent (e.g. methotrexate), a chemotherapeutic agent (e.g.epothilone B), a nitrogen mustard agent (e.g. bendamustine), or theactive agent or drug may be an siRNA.

In one set of embodiments, the payload is a drug or a combination ofmore than one drug. Such particles may be useful, for example, inembodiments where a targeting moiety may be used to direct a particlecontaining a drug to a particular localized location within a subject,e.g., to allow localized delivery of the drug to occur. Exemplarytherapeutic agents include chemotherapeutic agents such as doxorubicin(adriamycin), gemcitabine (gemzar), daunorubicin, procarbazine,mitomycin, cytarabine, etoposide, methotrexate, 5-fluorouracil (5-FU),vinca alkaloids such as vinblastine, vinoelbine, vindesine, orvincristine; bleomycin, taxanes such as paclitaxel (taxol) or docetaxel(taxotere), mTOR inhibitors such as sirolimus, temsirolimus, oreverolimus, aldesleukin, asparaginase, boronate esters or peptideboronic acid compounds such as bortezomib, busulfan, carboplatin,cladribine, camptothecin, CPT-11, 10-hydroxy-7-ethylcamptothecin (SN38),dacarbazine, S-I capecitabine, ftorafur, 5′deoxyfluorouridine, UFT,eniluracil, deoxycytidine, 5-azacytosine, 5-azadeoxycytosine,allopurinol, 2-chloroadenosine, trimetrexate, aminopterin,methylene-10-deazaminopterin (MDAM), oxaplatin, picoplatin, tetraplatin,satraplatin, platinum-DACH, ormaplatin, CI-973, JM-216, epirubicin,etoposide phosphate, 9-aminocamptothecin,10,11-methylenedioxycamptothecin, karenitecin, 9-nitrocamptothecin, TAS103, L-phenylalanine mustard, ifosphamidemefosphamide, perfosfamide,trophosphamide carmustine, semustine, bendamustine, epothilones A-E,tomudex, 6-mercaptopurine, 6-thioguanine, amsacrine, karenitecin,acyclovir, valacyclovir, ganciclovir, amantadine, rimantadine,lamivudine, zidovudine, bevacizumab, trastuzumab, rituximab, budesonide,and combinations thereof, or the therapeutic agent may be an siRNA.

In some embodiments, contemplated nanoparticles do not include a taxane(e.g. do not include docetaxel). In other embodiments, contemplatednanoparticles do not include a vinca alkaloid or a mTOR inhibitor.

Non-limiting examples of potentially suitable drugs include anti-canceragents, including, for example, docetaxel, mitoxantrone, andmitoxantrone hydrochloride. In another embodiment, the payload may be ananti-cancer drug such as 20-epi-1, 25 dihydroxyvitamin D3, 4-ipomeanol,5-ethynyluracil, 9-dihydrotaxol, abiraterone, acivicin, aclarubicin,acodazole hydrochloride, acronine, acylfiilvene, adecypenol, adozelesin,aldesleukin, all-tk antagonists, altretamine, ambamustine, ambomycin,ametantrone acetate, amidox, amifostine, aminoglutethimide,aminolevulinic acid, amrubicin, amsacrine, anagrelide, anastrozole,andrographolide, angiogenesis inhibitors, antagonist D, antagonist G,antarelix, anthramycin, anti-dorsalizdng morphogenetic protein-1,antiestrogen, antineoplaston, antisense oligonucleotides, aphidicolinglycinate, apoptosis gene modulators, apoptosis regulators, apurinicacid, ARA-CDP-DL-PTBA, arginine deaminase, asparaginase, asperlin,asulacrine, atamestane, atrimustine, axinastatin 1, axinastatin 2,axinastatin 3, azacitidine, azasetron, azatoxin, azatyrosine, azetepa,azotomycin, baccatin III derivatives, balanol, batimastat,benzochlorins, benzodepa, benzoylstaurosporine, beta lactam derivatives,beta-alethine, betaclamycin B, betulinic acid, BFGF inhibitor,bicalutamide, bisantrene, bisantrene hydrochloride,bisazuidinylspermine, bisnafide, bisnafide dimesylate, bistratene A,bizelesin, bleomycin, bleomycin sulfate, BRC/ABL antagonists, breflate,brequinar sodium, bropirimine, budotitane, busulfan, buthioninesulfoximine, cactinomycin, calcipotriol, calphostin C, calusterone,camptothecin derivatives, canarypox IL-2, capecitabine, caraceraide,carbetimer, carboplatin, carboxamide-amino-triazole,carboxyamidotriazole, carest M3, carmustine, earn 700, cartilage derivedinhibitor, carubicin hydrochloride, carzelesin, casein kinaseinhibitors, castanosperrnine, cecropin B, cedefingol, cetrorelix,chlorambucil, chlorins, chloroquinoxaline sulfonamide, cicaprost,cirolemycin, cisplatin, cis-porphyrin, cladribine, clomifene analogs,clotrimazole, collismycin A, collismycin B, combretastatin A4,combretastatin analog, conagenin, crambescidin 816, crisnatol, crisnatolmesylate, cryptophycin 8, cryptophycin A derivatives, curacin A,cyclopentanthraquinones, cyclophosphamide, cyclosporine, cycloplatam,cypemycin, cytarabine, cytarabine ocfosfate, cytolytic factor,cytostatin, dacarbazine, dacliximab, dactinomycin, daunorubicinhydrochloride, decitabine, dehydrodidemnin B, deslorelin, dexifosfamide,dexormaplatin, dexrazoxane, dexverapamil, dezaguanine, dezaguaninemesylate, diaziquone, didemnin B, didox, diethyhiorspermine,dihydro-5-azacytidine, dioxamycin, diphenyl spiromustine, docetaxel,docosanol, dolasetron, doxifluridine, doxorubicin, doxorubicinhydrochloride, droloxifene, droloxifene citrate, dromostanolonepropionate, dronabinol, duazomycin, duocannycin SA, ebselen, ecomustine,edatrexate, edelfosine, edrecolomab, eflomithine, eflomithinehydrochloride, elemene, elsarnitrucin, emitefur, enloplatin, enpromate,epipropidine, epirubicin, epirubicin hydrochloride, epristeride,erbulozole, erythrocyte gene therapy vector system, esorubicinhydrochloride, estramustine, estramustine analog, estramustine phosphatesodium, estrogen agonists, estrogen antagonists, etanidazole, etoposide,etoposide phosphate, etoprine, exemestane, fadrozole, fadrozolehydrochloride, fazarabine, fenretinide, filgrastim, finasteride,flavopiridol, flezelastine, floxuridine, fluasterone, fludarabine,fludarabine phosphate, fluorodaunorunicin hydrochloride, fluorouracil,fluorocitabine, forfenimex, formestane, fosquidone, fostriecin,fostriecin sodium, fotemustine, gadolinium texaphyrin, gallium nitrate,galocitabine, ganirelix, gelatinase inhibitors, gemcitabine, gemcitabinehydrochloride, glutathione inhibitors, hepsulfam, heregulin,hexamethylene bisacetamide, hydroxyurea, hypericin, ibandronic acid,idarubicin, idarubicin hydrochloride, idoxifene, idramantone,ifosfamide, ihnofosine, ilomastat, imidazoacridones, imiquimod,immunostimulant peptides, insulin-like growth factor-1 receptorinhibitor, interferon agonists, interferon alpha-2A, interferonalpha-2B, interferon alpha-N1, interferon alpha-N3, interferon beta-IA,interferon gamma-IB, interferons, interleukins, iobenguane,iododoxorubicin, iproplatm, irinotecan, irinotecan hydrochloride,iroplact, irsogladine, isobengazole, isohomohalicondrin B, itasetron,jasplakinolide, kahalalide F, lamellarin-N triacetate, lanreotide,lanreotide acetate, leinamycin, lenograstim, lentinan sulfate,leptolstatin, letrozole, leukemia inhibiting factor, leukocyte alphainterferon, leuprolide acetate, leuprolide/estrogen/progesterone,leuprorelin, levamisole, liarozole, liarozole hydrochloride, linearpolyamine analog, lipophilic disaccharide peptide, lipophilic platinumcompounds, lissoclinamide, lobaplatin, lombricine, lometrexol,lometrexol sodium, lomustine, lonidamine, losoxantrone, losoxantronehydrochloride, lovastatin, loxoribine, lurtotecan, lutetium texaphyrinlysofylline, lytic peptides, maitansine, mannostatin A, marimastat,masoprocol, maspin, matrilysin inhibitors, matrix metalloproteinaseinhibitors, maytansine, mechlorethamine hydrochloride, megestrolacetate, melengestrol acetate, melphalan, menogaril, merbarone,mercaptopurine, meterelin, methioninase, methotrexate, methotrexatesodium, metoclopramide, metoprine, meturedepa, microalgal protein kinaseC uihibitors, MIF inhibitor, mifepristone, miltefosine, mirimostim,mismatched double stranded RNA, mitindomide, mitocarcin, mitocromin,mitogillin, mitoguazone, mitolactol, mitomalcin, mitomycin, mitomycinanalogs, mitonafide, mitosper, mitotane, mitotoxin fibroblast growthfactor-saporin, mitoxantrone, mitoxantrone hydrochloride, mofarotene,molgramostim, monoclonal antibody, human chorionic gonadotrophin,monophosphoryl lipid a/myobacterium cell wall SK, mopidamol, multipledrug resistance gene inhibitor, multiple tumor suppressor 1-basedtherapy, mustard anticancer agent, mycaperoxide B, mycobacterial cellwall extract, mycophenolic acid, myriaporone, n-acetyldinaline,nafarelin, nagrestip, naloxone/pentazocine, napavin, naphterpin,nartograstim, nedaplatin, nemorubicin, neridronic acid, neutralendopeptidase, nilutamide, nisamycin, nitric oxide modulators, nitroxideantioxidant, nitrullyn, nocodazole, nogalamycin, n-substitutedbenzamides, O6-benzylguanine, octreotide, okicenone, oligonucleotides,onapristone, ondansetron, oracin, oral cytokine inducer, ormaplatin,osaterone, oxaliplatin, oxaunomycin, oxisuran, paclitaxel, paclitaxelanalogs, paclitaxel derivatives, palauamine, palmitoylrhizoxin,pamidronic acid, panaxytriol, panomifene, parabactin, pazelliptine,pegaspargase, peldesine, peliomycin, pentamustine, pentosan polysulfatesodium, pentostatin, pentrozole, peplomycin sulfate, perflubron,perfosfamide, perillyl alcohol, phenazinomycin, phenylacetate,phosphatase inhibitors, picibanil, pilocarpine hydrochloride,pipobroman, piposulfan, pirarubicin, piritrexim, piroxantronehydrochloride, placetin A, placetin B, plasminogen activator inhibitor,platinum complex, platinum compounds, platinum-triamine complex,plicamycin, plomestane, porfimer sodium, porfiromycin, prednimustine,procarbazine hydrochloride, propyl bis-acridone, prostaglandin J2,prostatic carcinoma antiandrogen, proteasome inhibitors, protein A-basedimmune modulator, protein kinase C inhibitor, protein tyrosinephosphatase inhibitors, purine nucleoside phosphorylase inhibitors,puromycin, puromycin hydrochloride, purpurins, pyrazorurin,pyrazoloacridine, pyridoxylated hemoglobin polyoxyethylene conjugate,RAF antagonists, raltitrexed, ramosetron, RAS farnesyl proteintransferase inhibitors, RAS inhibitors, RAS-GAP inhibitor, retelliptinedemethylated, rhenium RE 186 etidronate, rhizoxin, riboprine, ribozymes,RH retinarnide, RNAi, rogletimide, rohitukine, romurtide, roquinimex,rubiginone B1, ruboxyl, safingol, safingol hydrochloride, saintopin,sarcnu, sarcophytol A, sargramostim, SDI1 mimetics, semustine,senescence derived inhibitor 1, sense oligonucleotides, signaltransduction inhibitors, signal transduction modulators, simtrazene,single chain antigen binding protein, sizofuran, sobuzoxane, sodiumborocaptate, sodium phenylacetate, solverol, somatomedin bindingprotein, sonermin, sparfosafe sodium, sparfosic acid, sparsomycin,spicamycin D, spirogermanium hydrochloride, spiromustine, spiroplatin,splenopentin, spongistatin 1, squalamine, stem cell inhibitor, stem-celldivision inhibitors, stipiamide, streptonigrin, streptozocin,stromelysin inhibitors, sulfinosine, sulofenur, superactive vasoactiveintestinal peptide antagonist, suradista, suramin, swainsonine,synthetic glycosaminoglycans, talisomycin, tallimustine, tamoxifenmethiodide, tauromustine, tazarotene, tecogalan sodium, tegafur,tellurapyrylium, telomerase inhibitors, teloxantrone hydrochloride,temoporfin, temozolomide, teniposide, teroxirone, testolactone,tetrachlorodecaoxide, tetrazomine, thaliblastine, thalidomide,thiamiprine, thiocoraline, thioguanine, thiotepa, thrombopoietin,thrombopoietin mimetic, thymalfasin, thymopoietin receptor agonist,thymotrinan, thyroid stimulating hormone, tiazofurin, tin ethyletiopurpurin, tirapazamine, titanocene dichloride, topotecanhydrochloride, topsentin, toremifene, toremifene citrate, totipotentstem cell factor, translation inhibitors, trestolone acetate, tretinoin,triacetyluridine, triciribine, triciribine phosphate, trimetrexate,trimetrexate glucuronate, triptorelin, tropisetron, tubulozolehydrochloride, turosteride, tyrosine kinase inhibitors, tyrphostins, UBCinhibitors, ubenimex, uracil mustard, uredepa, urogenital sinus-derivedgrowth inhibitory factor, urokinase receptor antagonists, vapreotide,variolin B, velaresol, veramine, verdins, verteporfin, vinblastinesulfate, vincristine sulfate, vindesine, vindesine sulfate, vinepidinesulfate, vinglycinate sulfate, vinleurosine sulfate, vinorelbine orvinorelbine tartrate, vinrosidine sulfate, vinxaltine, vinzolidinesulfate, vitaxin, vorozole, zanoterone, zeniplatin, zilascorb,zinostatin, zinostatin stimalamer, or zorubicin hydrochloride.

In an embodiment, an active agent may (or may not be) conjugated to e.g.a disclosed hydrophobic polymer that forms part of a disclosednanoparticle, e.g an active agent may be conjugated (e.g. covalentlybound, e.g. directly or through a linking moiety) to PLA or PGLA, or aPLA or PLGA portion of a copolymer such as PLA-PEG or PLGA-PEG.

Preparation of Nanoparticles

In some embodiments, disclosed nanoparticles are formed by providing asolution comprising one or more polymers, and contacting the solutionwith a polymer nonsolvent to produce the particle. The solution may bemiscible or immiscible with the polymer nonsolvent. For example, awater-miscible liquid such as acetonitrile may contain the polymers, andparticles are formed as the acetonitrile is contacted with water, apolymer nonsolvent, e.g., by pouring the acetonitrile into the water ata controlled rate. The polymer contained within the solution, uponcontact with the polymer nonsolvent, may then precipitate to formparticles such as nanoparticles. Two liquids are said to be “immiscible”or not miscible, with each other when one is not soluble in the other toa level of at least 10% by weight at ambient temperature and pressure.Typically, an organic solution (e.g., dichloromethane, acetonitrile,chloroform, tetrahydrofuran, acetone, formamide, dimethylformamide,pyridines, dioxane, dimethysulfoxide, etc.) and an aqueous liquid (e.g.,water, or water containing dissolved salts or other species, cell orbiological media, ethanol, etc.) are immiscible with respect to eachother. For example, the first solution may be poured into the secondsolution (at a suitable rate or speed). In some cases, particles such asnanoparticles may be formed as the first solution contacts theimmiscible second liquid, e.g., precipitation of the polymer uponcontact causes the polymer to form nanoparticles while the firstsolution poured into the second liquid, and in some cases, for example,when the rate of introduction is carefully controlled and kept at arelatively slow rate, nanoparticles may form. The control of suchparticle formation can be readily optimized by one of ordinary skill inthe art using only routine experimentation.

In another embodiment, a nanoemulsion process is provided, such as theprocess represented in FIGS. 1, 2A, and 2B. For example, a therapeuticagent, a first polymer (for example, a diblock co-polymer such asPLA-PEG or PLGA-PEG, either of which may be optionally bound to aligand, e.g., GL2) and an optional second polymer (e.g., (PL(G)A-PEG orPLA), with an organic solution to form a first organic phase. Such firstphase may include about 5 to about 50% weight solids, e.g about 5 toabout 40% solids, or about 10 to about 30% solids. The first organicphase may be combined with a first aqueous solution to form a secondphase. The organic solution can include, for example, toluene, methylethyl ketone, acetonitrile, tetrahydrofuran, ethyl acetate, isopropylalcohol, isopropyl acetate, dimethylformamide, methylene chloride,dichloromethane, chloroform, acetone, benzyl alcohol, Tween 80, Span 80,or the like, and combinations thereof. In an embodiment, the organicphase may include benzyl alcohol, ethyl acetate, and combinationsthereof. The second phase can be between about 1 and 50 weight %, e.g.,about 5-40 weight %, solids. The aqueous solution can be water,optionally in combination with one or more of sodium cholate, ethylacetate, polyvinyl acetate and benzyl alcohol.

For example, the oil or organic phase may use a solvent that is onlypartially miscible with the nonsolvent (water). Therefore, when mixed ata low enough ratio and/or when using water pre-saturated with theorganic solvents, the oil phase remains liquid. The oil phase may beeemulsified into an aqueous solution and, as liquid droplets, shearedinto nanoparticles using, for example, high energy dispersion systems,such as homogenizers or sonicators. The aqueous portion of the emulsion,otherwise known as the “water phase”, may be surfactant solutionconsisting of sodium cholate and pre-saturated with ethyl acetate andbenzyl alcohol.

Emulsifying the second phase to form an emulsion phase may be performedin one or two emulsification steps. For example, a primary emulsion maybe prepared, and then emulsified to form a fine emulsion. The primaryemulsion can be formed, for example, using simple mixing, a highpressure homogenizer, probe sonicator, stir bar, or a rotor statorhomogenizer. The primary emulsion may be formed into a fine emulsionthrough the use of e.g., probe sonicator or a high pressure homogenizer,e.g., by using 1, 2, 3 or more passes through a homogenizer. Forexample, when a high pressure homogenizer is used, the pressure used maybe about 1000 to about 8000 psi, about 2000 to about 4000 psi 4000 toabout 8000 psi, or about 4000 to about 5000 psi, e.g., about 2000, 2500,4000 or 5000 psi.

Either solvent evaporation or dilution may be needed to complete theextraction of the solvent and solidify the particles. For better controlover the kinetics of extraction and a more scalable process, a solventdilution via aqueous quench may be used. For example, the emulsion canbe diluted into cold water to a concentration sufficient to dissolve allof the organic solvent to form a quenched phase. Quenching may beperformed at least partially at a temperature of about 5° C. or less.For example, water used in the quenching may be at a temperature that isless that room temperature (e.g., about 0 to about 10° C., or about 0 toabout 5° C.).

In some embodiments, not all of the therapeutic agent (e.g., docetaxel)is encapsulated in the particles at this stage, and a drug solubilizeris added to the quenched phase to form a solubilized phase. The drugsolubilizer may be for example, Tween 80, Tween 20, polyvinylpyrrolidone, cyclodextran, sodium dodecyl sulfate, or sodium cholate.For example, Tween-80 may added to the quenched nanoparticle suspensionto solubilize the free drug and prevent the formation of drug crystals.In some embodiments, a ratio of drug solubilizer to therapeutic agent(e.g., docetaxel) is about 100:1 to about 10:1.

The solubilized phase may be filtered to recover the nanoparticles. Forexample, ultrafiltration membranes may be used to concentrate thenanoparticle suspension and substantially eliminate organic solvent,free drug, and other processing aids (surfactants). Exemplary filtrationmay be performed using a tangential flow filtration system. For example,by using a membrane with a pore size suitable to retain nanoparticleswhile allowing solutes, micelles, and organic solvent to pass,nanoparticles can be selectively separated. Exemplary membranes withmolecular weight cut-offs of about 300-500 kDa (˜5-25 nm) may be used.

Diafiltration may be performed using a constant volume approach, meaningthe diafiltrate (cold deionized water, e.g., about 0 to about 5° C., or0 to about 10° C.) may added to the feed suspension at the same rate asthe filtrate is removed from the suspension. In some embodiments,filtering may include a first filtering using a first temperature ofabout 0 to about 5° C., or 0 to about 10° C., and a second temperatureof about 20 to about 30° C., or 15 to about 35° C. For example,filtering may include processing about 1 to about 6 diavolumes at about0 to about 5° C., and processing at least one diavolume (e.g., about 1to about 3 or about 1-2 diavolumes) at about 20 to about 30° C.

After purifying and concentrating the nanoparticle suspension, theparticles may be passed through one, two or more sterilizing and/ordepth filters, for example, using ˜0.2 μm depth pre-filter.

In another embodiment of preparing nanoparticles, an organic phase isformed composed of a mixture of a therapeutic agent, e.g., docetaxel,and polymer (homopolymer, co-polymer, and co-polymer with ligand). Theorganic phase is mixed with an aqueous phase at approximately a 1:5ratio (oil phase:aqueous phase) where the aqueous phase is composed of asurfactant and some dissolved solvent. The primary emulsion is formed bythe combination of the two phases under simple mixing or through the useof a rotor stator homogenizer. The primary emulsion is then formed intoa fine emulsion through the use of a high pressure homogenizer. The fineemulsion is then quenched by addition to deionized water under mixing.The quench:emulsion ratio is approximately 8.5:1. Then a solution ofTween (e.g., Tween 80) is added to the quench to achieve approximately2% Tween overall. This serves to dissolve free, unencapsulated drug. Thenanoparticles are then isolated through either centrifugation orultrafiltration/diafiltration.

It will be appreciated that the amounts of polymer and therapeutic oractive agent that are used in the preparation of the formulation maydiffer from a final formulation. For example, some active agent may notbecome completely incorporated in a nanoparticle and such freetherapeutic agent may be e.g., filtered away.

Pharmaceutical Compositions

Nanoparticles disclosed herein may be combined with pharmaceuticalacceptable carriers to form a pharmaceutical composition, according toanother aspect of the disclosure. As would be appreciated by one ofskill in this art, the carriers may be chosen based on the route ofadministration as described below, the location of the target issue, thedrug being delivered, the time course of delivery of the drug, etc.

The pharmaceutical compositions of this disclosure can be administeredto a patient by any means known in the art including oral and parenteralroutes. The term “patient,” as used herein, refers to humans as well asnon-humans, including, for example, mammals, birds, reptiles,amphibians, and fish. For instance, the non-humans may be mammals (e.g.,a rodent, a mouse, a rat, a rabbit, a monkey, a dog, a cat, a primate,or a pig). In certain embodiments parenteral routes are desirable sincethey avoid contact with the digestive enzymes that are found in thealimentary canal. According to such embodiments, inventive compositionsmay be administered by injection (e.g., intravenous, subcutaneous orintramuscular, intraperitoneal injection), rectally, vaginally,topically (as by powders, creams, ointments, or drops), or by inhalation(as by sprays).

In a particular embodiment, the nanoparticles of the present disclosureare administered to a subject in need thereof systemically, e.g.,parenterally, or by IV infusion or injection.

In some embodiments, a composition suitable for freezing iscontemplated, including nanoparticles disclosed herein and a solutionsuitable for freezing, e.g., a sucrose and/or a salt solution is addedto the nanoparticle suspension. The sucrose may act, e.g., as acryoprotectant to prevent the particles from aggregating upon freezing.For example, provided herein is a nanoparticle formulation comprising aplurality of disclosed nanoparticles, sucrose, an ionic halide, andwater; wherein the nanoparticles/sucrose/water is about3-30%/10-30%/50-90% (w/w/w) or about 5-10%/10-15%/80-90% (w/w/w). Forexample, such solution may include nanoparticles as disclosed herein,about 5% to about 20% by weight sucrose and an ionic halide such assodium chloride, in a concentration of about 10-100 mM.

Compositions and Methods of Treatment

Nanoparticles disclosed herein may be combined with pharmaceuticalacceptable carriers to form a pharmaceutical composition. As would beappreciated by one of skill in this art, the carriers may be chosenbased on the route of administration as described below, the location ofthe target issue, the drug being delivered, the time course of deliveryof the drug, etc.

The pharmaceutical compositions and particles disclosed herein can beadministered to a patient by any means known in the art including oraland parenteral routes. The term “patient,” as used herein, refers tohumans as well as non-humans, including, for example, mammals, birds,reptiles, amphibians, and fish. For instance, the non-humans may bemammals (e.g., a rodent, a mouse, a rat, a rabbit, a monkey, a dog, acat, a primate, or a pig). In certain embodiments parenteral routes aredesirable since they avoid contact with the digestive enzymes that arefound in the alimentary canal. According to such embodiments, inventivecompositions may be administered by injection (e.g., intravenous,subcutaneous or intramuscular, intraperitoneal injection), rectally,vaginally, topically (as by powders, creams, ointments, or drops), or byinhalation (as by sprays).

In a particular embodiment, disclosed nanoparticles may be administeredto a subject in need thereof systemically, e.g., by IV infusion orinjection.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P., and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Inone embodiment, the inventive conjugate is suspended in a carrier fluidcomprising 1% (w/v) sodium carboxymethyl cellulose and 0.1% (v/v) TWEEN™80. The injectable formulations can be sterilized, for example, byfiltration through a bacteria-retaining filter, or by incorporatingsterilizing agents in the form of sterile solid compositions which canbe dissolved or dispersed in sterile water or other sterile injectablemedium prior to use.

Solid dosage forms for oral administration include capsules, tablets,pills, powders, and granules. In such solid dosage forms, theencapsulated or unencapsulated conjugate is mixed with at least oneinert, pharmaceutically acceptable excipient or carrier such as sodiumcitrate or dicalcium phosphate and/or (a) fillers or extenders such asstarches, lactose, sucrose, glucose, mannitol, and silicic acid, (b)binders such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinylpyrrolidinone, sucrose, and acacia, (c) humectantssuch as glycerol, (d) disintegrating agents such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates,and sodium carbonate, (e) solution retarding agents such as paraffin,(f) absorption accelerators such as quaternary ammonium compounds, (g)wetting agents such as, for example, cetyl alcohol and glycerolmonostearate, (h) absorbents such as kaolin and bentonite clay, and (i)lubricants such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate, and mixtures thereof. Inthe case of capsules, tablets, and pills, the dosage form may alsocomprise buffering agents.

Disclosed nanoparticles may be formulated in dosage unit form for easeof administration and uniformity of dosage. The expression “dosage unitform” as used herein refers to a physically discrete unit ofnanoparticle appropriate for the patient to be treated. For anynanoparticle, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. An animal model may also be used toachieve a desirable concentration range and route of administration.Such information can then be used to determine useful doses and routesfor administration in humans. Therapeutic efficacy and toxicity ofnanoparticles can be determined by standard pharmaceutical procedures incell cultures or experimental animals, e.g., ED₅₀ (the dose istherapeutically effective in 50% of the population) and LD₅₀ (the doseis lethal to 50% of the population). The dose ratio of toxic totherapeutic effects is the therapeutic index, and it can be expressed asthe ratio, LD₅₀/ED₅₀. Pharmaceutical compositions which exhibit largetherapeutic indices may be useful in some embodiments. The data obtainedfrom cell culture assays and animal studies can be used in formulating arange of dosage for human use.

In an exemplary embodiment, a pharmaceutical composition is disclosedthat includes a plurality of nanoparticles each comprising a therapeuticagent and a pharmaceutically acceptable excipient.

In some embodiments, a composition suitable for freezing iscontemplated, including nanoparticles disclosed herein and a solutionsuitable for freezing, e.g., a sugar (e.g. sucrose) solution is added toa nanoparticle suspension. The sucrose may, e.g., act as acryoprotectant to prevent the particles from aggregating upon freezing.For example, provided herein is a nanoparticle formulation comprising aplurality of disclosed nanoparticles, sucrose and water; wherein, forexample, the nanoparticles/sucrose/water are present at about5-10%/10-15%/80-90% (w/w/w).

In some embodiments, therapeutic particles disclosed herein may be usedto treat, alleviate, ameliorate, relieve, delay onset of, inhibitprogression of, reduce severity of, and/or reduce incidence of one ormore symptoms or features of a disease, disorder, and/or condition. Forexample, disclosed therapeutic particles, that include taxane, e.g.,docetaxel, may be used to treat cancers such as breast, lung, orprostate cancer in a patient in need thereof. Other types of tumors andcancer cells to be treated with therapeutic particles of the presentinvention include all types of solid tumors, such as those which areassociated with the following types of cancers: lung, squamous cellcarcinoma of the head and neck (SCCHN), pancreatic, colon, rectal,esophageal, prostate, breast, ovarian carcinoma, renal carcinoma,lymphoma and melanoma. The tumor can be associated with cancers of(i.e., located in) the oral cavity and pharynx, the digestive system,the respiratory system, bones and joints (e.g., bony metastases), softtissue, the skin (e.g., melanoma), breast, the genital system, theurinary system, the eye and orbit, the brain and nervous system (e.g.,glioma), or the endocrine system (e.g., thyroid) and is not necessarilythe primary tumor. Tissues associated with the oral cavity include, butare not limited to, the tongue and tissues of the mouth. Cancer canarise in tissues of the digestive system including, for example, theesophagus, stomach, small intestine, colon, rectum, anus, liver, gallbladder, and pancreas. Cancers of the respiratory system can affect thelarynx, lung, and bronchus and include, for example, non-small cell lungcarcinoma. Tumors can arise in the uterine cervix, uterine corpus, ovaryvulva, vagina, prostate, testis, and penis, which make up the male andfemale genital systems, and the urinary bladder, kidney, renal pelvis,and ureter, which comprise the urinary system.

Disclosed methods for the treatment of cancer (e.g. breast or prostatecancer) may comprise administering a therapeutically effective amount ofthe disclosed therapeutic particles to a subject in need thereof, insuch amounts and for such time as is necessary to achieve the desiredresult. In certain embodiments of the present invention a“therapeutically effective amount” is that amount effective fortreating, alleviating, ameliorating, relieving, delaying onset of,inhibiting progression of, reducing severity of, and/or reducingincidence of one or more symptoms or features of e.g. a cancer beingtreated.

In some embodiments, disclosed therapeutic nanoparticles that includeepothilone, e.g. epothilone B may be used to treat cancers such asbreast, prostate, colon, glioblastoma, acute lymphoblastic leukemia,osteosarcoma, non-Hodkgin's lymphoma, or lung cancer such a non-smallcell lung cancer, in a patient in need thereof.

In other embodiments, disclosed therapeutic nanoparticles that include acorticosteroid such as budesonide may be used to treat asthma,osteoarthritis, dermatitis, and inflammatory disorders such asinflammatory bowel disease, ulcerative colitis, and/or Crohn's disease(treatment of cancers such as colon cancer is also contemplated.)

Drug Release Rate

This disclosure relates in part to a method for predicting andcontrolling the rate of drug release from nanoparticles by measurementof the glass transition temperature (T_(g)) of drug loaded nanoparticlesin aqueous suspension. For example, measurement under suspensionconditions may be needed to manipulate the chemical composition andphysical properties of the nanoparticle in the blood stream upon IVadministration. Nanoparticle formulations can also be designed toexhibit desired drug release rates based upon the T_(g) ofnanoparticles.

The glass transition temperature (T_(g)) of drug bearing nanoparticlesin aqueous suspension may be an indicator of the drug releasecharacteristics of the nanoparticle. Identification of nanoparticlesuspension T_(g) allows for its use as a predictor of drug releaseproperties, enables rational design of nano-particulate polymer and drugformulations that possess desired drug release rates, and additionallyallows rapid screening of formulations to identify high value targetsfor further investigation.

Forming nanoparticles using an emulsion process typically yields anamorphous solid dispersion of drug in polymer. Depending on its chemicalstructure, the polymer may partially crystallize; however, often thelack of stereo-regularity of repeating units along the polymer chaincauses the polymer to adopt an amorphous state. This rigid glassy stateis similar to that obtained by cooling a polymer melt. The melt phase ischaracterized by rubber like properties. The transition to the glassystate is accompanied by changes in the polymer material propertiesincluding hardness, Young's modulus, and heat capacity. Severaltechniques that monitor changes in these properties can be used todetermine the temperature (or temperature range) over which this rubberto glass transition, known as the glass transition temperature (T_(g)),occurs. The T_(g) can be, for example, dependent upon the purity of thepolymer. For example, the presence of small molecules, such as drugmolecules or active agents, solvent or non-solvent molecules (i.e.,water), in nanoparticles can affect the T_(g) of the polymer, e.g., highsurface to volume ratio of nanoparticles may contribute to the watercontent of the polymer phase. The T_(g) of polymeric nanoparticles in anaqueous suspension may be distinctly different from that of a mesoscopicpolymer-drug mixture of the same chemical composition or that ofdrug-bearing nanoparticles in dry powder form.

Drug within the nanoparticles may be molecularly dispersed or may formnanocrystals of dimensions smaller than those of the polymernanoparticle. In both instances, the diffusional-based release of drugfrom the nanoparticle is dependent upon its transport through thepolymer matrix and into the surrounding aqueous phase. As such, thedrug's intrinsic solubility in the polymer matrix, its diffusioncoefficient, the diffusion path length, viscosity as well as temperatureof the polymer matrix can be factors in the rate of drug release fromthe nanoparticles. For example, upon intravenous injection,nanoparticles may quickly equilibrate to physiological temperature (37°C.) and therefore, the material properties of the polymer matrix at thistemperature can influence the rate of drug release. Drug diffusionacross the polymer and its release from the nanoparticle can be slowerif the polymeric matrix is in a rigid glassy state at 37° C., andrelatively faster if the polymer matrix is in a rubbery state at thistemperature. That is, under physiological conditions, nanoparticles thatpossess a T_(g) at or below 37° C. may release drug at a faster ratethan those possessing a T_(g) above 37° C.

For example, nanoparticles comprising PLA-PEG and low molecular weightPLA (e.g., 6.5 kDa PLA) have a lower T_(g) than nanoparticles comprisingPLA-PEG alone. Addition of high molecular weight PLA (e.g., 75 kDa PLA)to nanoparticles containing PLA-PEG raises the T_(g) above that ofnanoparticles comprising PLA-PEG alone. By varying the type and amountof homopolymer, such as PLA, in a nanoparticle composition, the T_(g)can be altered which then directly affects the rate of drug release fromthe nanoparticles. Similarly, the Tg of the nanoparticles that includediblock copolymers (e.g. PLA-PEG) only is typically a function of themlar mas of the core forming block (e.g. PLA). Glass transitiontemperatures of the polymeric components of nanoparticles may be used toselect compositions that impart a range of thermal characteristics toparticles and enables prediction of their drug release properties.

This disclosure relates in part to methods for screening polymer anddrug systems to identify combinations that have the necessary thermalcharacteristics (glass transition temperatures) associated with adesired drug release profile. By assaying the T_(g) of a givennanoparticle composition, the drug release rate of the nanoparticles canbe predicted. Screening based on T_(g), or T_(b), the temperature atwhich the nanoparticle glass transition begins, can rapidly identifynanoparticle polymer combinations that release drug at a desired rate.In combination with a high throughput means of nanoparticle fabrication,this disclosure allows for rapid screening of polymer and drugcombinations to arrive at a select number of systems that can then besubjected to traditional more detailed drug release studies.

For example, analysis of drug loaded nanoparticles by DifferentialScanning calorimetry (DSC) under aqueous suspension conditionsdemonstrates the correlation between drug release rate and nanoparticleglass transition temperature. DSC is a technique in which the heat flowrate into a substance and a reference is measured as a function oftemperature while the substance and reference are subjected to acontrolled temperature program. DSC techniques include Heat Flux DSC andPower Compensation DSC. In Heat Flux DSC, the instrument consists of asingle cell containing reference and sample holders separated by abridge that acts as a heat-leak. This assembly sits within a heatingblock or furnace that is a constant temperature body. Thermocouples inthermal contact with the sample and reference platforms measure thetemperatures of the sample pan and reference pan while the heating blocktemperature is increased (heating cycle) or decreased (cooling cycle) ata given rate, for example 10° C./minute. The experimental outputconsists of the temperature differential (or heat flow differential inWatt/gram) between the sample and reference pans plotted against theheating block temperature. In contrast, in Power Compensation DSC, theinstrument consists of two separate but identical furnaces, one housingthe sample and the other the reference. In this case, the powerdifferential necessary to maintain the two furnaces at constanttemperature serves as the basis for differential heat flow. Theexperimental output is analogous to that in Heat Flux DSC.

The glass transition temperatures of nanoparticles in aqueous suspensioncan also be determined using Modulated Differential Scanning calorimetry(MDSC). In this technique, a sinusoidal temperature modulation isoverlaid on a linear heating or cooling rate. This is combined with amathematical procedure separates the total heat flow (similar to what isobserved in conventional DSC) into reversing and non-reversing heat flowcomponents. The reversing heat flow component derives from the heatcapacity of the sample as well as events that respond directly tochanges in the ramp rate and are reversing at the time and temperatureat which they are observed. In other words, events (e.g., glasstransition temperature) that are fast enough to be reversing on the timescale of the sinusoidal temperature modulation will contribute to thissignal. Events that do not respond to changes in ramp rate (e.g.,enthalpic relaxation at the glass transition temperature) are observedin the non-reversing heat flow component. By separating the reversingand non-reversing heat flow components of the total heat flow, MDSCallows the separation of glass transition events from enthalpicrelaxation events. Thus, unlike conventional DSC, with MDSC accuratedetermination of the glass transition temperature of samples thatexhibit strong enthalpic relaxations at the glass transition temperaturebecomes possible. For example, when nanoparticle suspensions areanalyzed using MDSC and the reversing heat flow component plottedagainst sample temperature, the curves may appear similar to thoseobserved in conventional DSC (total heat flow curves versus sampletemperature), confirming that the transitions observed by conventionalDSC are glass transition events rather than ethalpic relaxations.

The rate of drug release can be measured using an in vitro dissolution(i.e., suspension and centrifugation) technique. Nanoparticles aresuspended in a release medium, such as hydroxypropyl βCD (Trapsol) inPBS. After a period of time, the suspension is centrifuged, and a sampleof the medium from the upper part of the centrifuged suspension iswithdrawn without causing turbulence to avoid re-suspending thenanoparticle pellet at the centrifuge tube bottom. The sample is thenanalyzed by HPLC to determine the amount of drug released from thenanoparticles.

In one aspect, the disclosure provides a pharmaceutical aqueoussuspension comprising a plurality of nanoparticles, having a glasstransition temperature between about 37° C. and about 50° C., whereineach of the nanoparticles comprises a therapeutic agent and a blockcopolymer having at least one hydrophobic portion and at least onehydrophilic portion.

The hydrophobic portion may be selected from poly(D,L-lactic) acid andpoly(lactic acid-co-glycolic acid). The hydrophilic portion may bepoly(ethylene)glycol. The nanoparticles may further comprisepoly(D,L-lactic) acid or poly(lactic) acid-co-poly(glycolic) acid.

In one embodiment, the nanoparticles may comprise about 0.2 to about 35weight percent of a therapeutic agent; about 10 to about 99 weightpercent poly(D,L-lactic) acid-block-poly(ethylene)glycol copolymer orpoly(lactic)-co-poly(glycolic) acid-block-poly(ethylene)glycolcopolymer; and about 0 to about 50 or 0 to about 75 weight percentpoly(D,L-lactic) acid or poly(lactic) acid-co-poly(glycolic) acid. Inanother embodiment, the poly(D,L-lactic) acid portion of the copolymerhas a number average molecular weight of about 16 kDa, and thepoly(ethylene)glycol portion of the copolymer has a number averagemolecular weight of about 5 kDa. In another embodiment, thepoly(D,L-lactic) acid portion of the copolymer has a number averagemolecular weight of about 50 kDa, and the poly(ethylene)glycol portionof the copolymer has a number average molecular weight of about 5 kDa.In an embodiment, the poly(D,L-lactic) acid has a number averagemolecular weight of about 6.5 kDa. In another embodiment, thepoly(D,L-lactic) acid has a number average molecular weight of about 75kDa.

In one embodiment, a provided aqueous suspension of nanoparticles has aglass transition temperature may be about 37° C. to about 39° C., 37° C.to about 39.5° C., 39.5° C. to about 41° C., about 42° C. to about 50°C., or about 42° C. to about 44° C. In another embodiment, an aqueoussuspension of nanoparticles may have a glass transition temperature thatmay be about 37° C. to about 38° C. In some embodiments, providednanoparticles or suspensions do not include nanoparticles or suspensionshaving a glass transition temperature of 40° C., or 39.5° C. to about41° C. The term “about” in the context of glass transition temperaturegenerally means±0.5° C. The glass transition temperature may be measuredby Heat Flux Differential Scanning calorimetry, Power CompensationDifferential Scanning calorimetry, and/or Modulated DSC.

In one embodiment, disclosed nanoparticles release less than about 50%of the therapeutic agent as determined in an in vitro dissolution testat a 4 hour time point. In another embodiment, the nanoparticles releasebetween about 50 to about 70% of the therapeutic agent as determined inan in vitro dissolution test at a 4 hour time point. In anotherembodiment, the nanoparticles release between about 70 to about 100% ofthe therapeutic agent as determined in an in vitro dissolution test at a4 hour time point.

In another aspect, the disclosure provides a method for determining thedrug release rate of a therapeutic polymeric nanoparticle composition,comprising:

a) providing at least one first plurality of polymeric nanoparticlescomprising a first therapeutic agent, a first block copolymer having atleast one hydrophobic portion and at least one hydrophilic portion, andoptionally poly(D,L-lactic) acid or poly(lactic) acid-co-poly(glycolic)acid;

b) determining the nanoparticle glass transition temperature for the atleast one first plurality of polymeric nanoparticles;

c) determining the drug release rate from the at least one firstplurality of polymeric nanoparticles; and

d) determining the correlation between the nanoparticle glass transitiontemperature and the drug release rate for the at least one firstplurality of polymeric nanoparticles.

In an embodiment, the method for determining the drug release rate of atherapeutic polymeric nanoparticle composition further comprises:

e) providing at least one second plurality of polymeric nanoparticlescomprising a second therapeutic agent, a second block copolymer havingat least one hydrophobic portion and at least one hydrophilic portion,and optionally poly(D,L-lactic) acid or poly(lactic)acid-co-poly(glycolic) acid, wherein the second therapeutic agent andsecond block copolymer may be the same or different as the firsttherapeutic agent and first block copolymer;

f) determining the nanoparticle glass transition temperature for the atleast one second plurality of polymeric nanoparticles; and

g) predicting the drug release rate for the at least one secondplurality of polymeric nanoparticles based on the nanoparticle glasstransition temperature for the at least one second plurality ofpolymeric nanoparticles and the correlation determined in step d).

In another embodiment, the method for determining the drug release rateof a therapeutic polymeric nanoparticle composition further comprisesconfirming the predicted drug release rate from the at least one secondplurality of polymeric nanoparticles using an in vitro dissolution test.

The first and/or second therapeutic agent may be a taxane agent, such asdocetaxel.

In one embodiment, the hydrophobic portions of the first and secondblock copolymers can each be selected from poly(D,L-lactic) acid andpoly(lactic acid-co-glycolic acid). In an embodiment, the hydrophilicportions of the first and second block copolymers can each bepoly(ethylene)glycol. In one embodiment, the first and/or second blockcopolymer may comprise about 0.2 to about 35 weight percent of atherapeutic agent; about 10 to about 99 weight percent poly(D,L-lactic)acid-block-poly(ethylene)glycol copolymer orpoly(lactic)-co-poly(glycolic) acid-block-poly(ethylene)glycolcopolymer; and about 0 to about 50 weight percent poly(D,L-lactic) acidor poly(lactic) acid-co-poly(glycolic) acid. In an embodiment, thepoly(D,L-lactic) acid portion of the first and/or second block copolymermay have a number average molecular weight of about 16 kDa or about 50kDa, and the poly(ethylene)glycol portion of the first and/or secondblock copolymer may have a weight average molecular weight of about 5kDa. In another embodiment, the poly(D,L-lactic) acid may have a numberaverage molecular weight of about 8.5 kDa. In one embodiment, thepoly(D,L-lactic) acid may have a number average molecular weight ofabout 75 kDa. In an embodiment, the poly(D,L-lactic) acid portion of thefirst and/or second block copolymer may have a number average molecularweight of about 50 kDa, and the poly(ethylene)glycol portion of thefirst and/or second block copolymer may have a number average molecularweight of about 5 kDa.

Also provided herein is a method for screening nanoparticle suspensions(e.g. to identify a predetermined release rate of a therapeutic agent),comprising:

i) providing a suspension comprising a first plurality of polymericnanoparticles, wherein the nanoparticles each comprise a therapeuticagent, a block copolymer having at least one hydrophobic portion and atleast one hydrophilic portion, and a homopolymer selected frompoly(D,L-lactic) acid or poly(lactic) acid-co-poly(glycolic) acid;

ii) determining the glass transition temperature for the suspension;

iii) increasing or decreasing the amount of the homopolymer in the firstplurality of polymeric nanoparticles; and

iv) repeating steps i)-iii) until a suspension with a desired glasstransition temperature is achieved.

For example, provided herein is a method for screening nanoparticlesuspensions to identify a suspension having a specific release rate,comprising:

a) separately preparing a plurality of suspensions having nanoparticlescomprising a therapeutic agent, a block copolymer having at least onehydrophobic portion and at least one hydrophilic portion, and optionallya homopolymer selected from poly(D,L-lactic) acid or poly(lactic)acid-co-poly(glycolic) acid; wherein each suspension is in a separatecompartment, each suspension comprises a pre-determined molecular weightof the block copolymer and if present, a pre-determined molecular weightof the homopolymer;

b) determining the glass transition temperature of each of thesuspensions;

c) identifying the suspension having a pre-determined glass transitiontemperature.

EXAMPLES

The invention now being generally described, it will be more readilyunderstood by reference to the following examples which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention inany way.

Example 1 Preparation of PLA-PEG

The synthesis is accomplished by ring opening polymerization ofd,l-lactide with α-hydroxy-ω-methoxypoly(ethylene glycol) as themacro-initiator, and performed at an elevated temperature using Tin (II)2-Ethyl hexanoate as a catalyst, as shown below (PEG Mn≈5,000 Da; PLAMn≈16,000 Da; PEG-PLA M_(n)≈21,000 Da)

The polymer is purified by dissolving the polymer in dichloromethane,and precipitating it in a mixture of hexane and diethyl ether. Thepolymer recovered from this step shall be dried in an oven.

Example 2 Nanoparticle Preparation—Emulsion Process

An organic phase is formed composed of a mixture of docetaxel (DTXL) andpolymer (homopolymer, co-polymer, and/or co-polymer with ligand). Theorganic phase is mixed with an aqueous phase at approximately a 1:5ratio (oil phase:aqueous phase) where the aqueous phase is composed of asurfactant and some dissolved solvent. In order to achieve high drugloading, about 30% solids in the organic phase is used.

The primary, coarse emulsion is formed by the combination of the twophases under simple mixing (stir bar) or through the use of a rotorstator homogenizer. The primary emulsion is then formed into a fineemulsion through the use of a high pressure homogenizer. Generally 1-3passes through a 100 micron Z-chamber at 9000 psig is used to produce anemulsion with the target particle size.

The fine emulsion is then quenched by addition to deionized water at agiven temperature under mixing. In the quench unit operation, theemulsion is added to a cold aqueous quench under agitation. This servesto extract a significant portion of the oil phase solvents, effectivelyhardening the nanoparticles for downstream filtration. Thequench:emulsion ratio is approximately 5:1.

A solution of 35% (wt %) of Tween 80 is added to the quench to achieveapproximately 4% Tween 80 overall After the emulsion is quenched asolution of Tween-80 is added which acts as a drug solubilizer, allowingfor effective removal of unencapsulated drug during filtration.

The temperature must remain cold enough with a dilute enough suspension(low enough concentration of solvents) to remain below the T_(g) of theparticles. If the Q:E ratio is not high enough, then the higherconcentration of solvent plasticizes the particles and allows for drugleakage. Conversely, colder temperatures allow for high drugencapsulation at low Q:E ratios (to ˜3:1), making it possible to run theprocess more efficiently.

The nanoparticles are then isolated through a tangential flow filtrationprocess to concentrate the nanoparticle suspension and buffer exchangethe solvents, unencapsulated drug, and drug solubilizer from the quenchsolution into water. A regenerated cellulose membrane is used with amolecular weight cutoffs (MWCO) of 300. A constant volume diafiltration(DF) is performed to remove the quench solvents, free drug and Tween-80.To perform a constant-volume DF, buffer is added to the retentate vesselat the same rate the filtrate is removed.

The filtered nanoparticle slurry is then thermal cycled to an elevatedtemperature during workup. A small portion (typically 5-10%) of theencapsulated drug is released from the nanoparticles very quickly afterits first exposure to 25° C. Because of this phenomenon, batches thatare held cold during the entire workup are susceptible to free drug ordrug crystals forming during delivery or any portion of unfrozenstorage.

After the filtration process the nanoparticle suspension is passedthrough a sterilizing grade filter (0.2 μm absolute). Pre-filters areused to protect the sterilizing grade filter in order to use areasonable filtration area/time for the process.

The filters normally used are Pall SXMPDD1404 (KS50P/EKSP double layer,(0.1-0.3 μm Nominal); Pall Life Sciences Supor EKV 0.65/0.2 micronsterilizing grade PES filter.

0.2 m² of filtration surface area per kg of nanoparticles for depthfilters and 1.3 m² of filtration surface area per kg of nanoparticlesfor the sterilizing grade filters can be used.

Example 3 Differential Scanning calorimetry of Polymers

The glass transition temperatures of the nanoparticles and theirpolymeric components were measured using either a TA Instruments Q200 ora TA Instruments Q2000 Heat Flux DSC equipped with the Tzero (T_(o))technology. The sample pan (containing either 20-70 μL nanoparticlesuspension or 3-10 mg polymer) and reference pans (comprised of an emptysample pan) were heated between 4° C. and 70° C. at a ramp rate of 10°C. (or 20° C.) per minute. High purity dry nitrogen was used to purgethe furnace during analysis.

In conventional Heat Flux DSC, two thermocouples, one each for thesample and reference pans are used. When the furnace temperature isramped at a constant heating rate (for example 10° C. per minute), thesample temperature (T_(s)) lags behind the reference temperature (T_(r))to an extent equal to the heat capacity of the sample provided thesample and reference pan masses are identical. The temperaturedifference, ΔT=T_(s)−T_(r), is recorded as a function of furnace blocktemperature. Typically, the instrument output consists of the heat flowrate per unit mass plotted against heating block temperature. The heatflow rate per unit mass in Watt/g (y-axis), consists of ΔT/R, where R isthe thermal resistance of the Constantan bridge that connects (andsupports) the reference and sample pans. Thus, when the experiment isstarted, the DSC signal shifts from zero to a steady state value(T_(s)−T_(r))/R, establishing the experimental baseline. When a glasstransition is encountered either on a heating cycle or cooling cycle,the steady state value shifts as a result of increase in the sample heatcapacity above the sample T_(g). Typically, the temperature at halfheight of the heat capacity change, ½ΔCp is defined as the sample T_(g).

In Heat Flux DSC's equipped with T₀ technology, a third thermocouplemeasures the temperature (T₀) of the Constantan bridge that connects(and supports) the reference and sample pans and the heat flow equationconsists of three additional terms, one to account for the differentresistances of the sample and reference cells and one each for thedifferences in thermal capacitance and heating rates. All DSC datapresented below was acquired on instruments equipped with T₀ technology.Additionally, the data was processed using the “T4 mode”, i.e., usingthe four term heat flow equation. For some experiments, the “T4P mode”was used. In this mode, a correction for differences between sample andreference pan weights was used in addition to those described for the T4mode.

Solid polymer samples, including poly(D,L-lactide)-block-poly(ethyleneglycol) (PLA-PEG, Mn PLA block=16 kDa; Mn PEG block=5 kDa),poly(D,L-lactide) (PLA, Mn=6 kDa), and poly(D,L-lactide) (PLA, Mn=75kDa) were analyzed using hermetically sealed pans, and instrumentconstants were determined using the T4 mode. The DSC results obtainedare shown in FIGS. 3 through 7 where the glass transition temperaturewas assigned using the point of inflexion method (T_(g)=point of largestslope).

FIG. 3 shows the DSC curve of poly(D,L-lactide)-block-poly(ethyleneglycol) (PLA-PEG, Mn PLA block=16 kDa; Mn PEG block=5 kDa) whenrecovered from a melt polymerization and having been cooled at anunknown cooling rate. The DSC measurement was repeated. The top curve isthe first heat, and the bottom curve is the second heat. Two distincttransitions are observed in the DSC curve of PLA-PEG (16 kDa-5 kDa)block copolymer. The transition between 1 and 10° C. is a T_(g) of themixed PEG and PLA phases, while the transition between 40 and 50° C. isa melting peak of a PEG rich phase. Homopolymer PEG of similar molarmasses typically exhibit T_(g)'s between −50 and −25° C., while PLAhomopolymer of similar molar mass exhibits a T_(g) in the 30-50° C.range. The observed T_(g) lies between that expected for a pure PEGhomopolymer and a pure PLA homopolymer indicating that this copolymersample is comprised of a mixed phase containing both PEG and PLA.

FIG. 4A shows the DSC curve of poly(D,L-lactide)-block-poly(ethyleneglycol) (PLA-PEG, Mn PLA block=16 kDa; Mn PEG block=5 kDa) whenrecovered from a precipitation of polymer solution (100 mg/mL indichloromethane) into a binary non-solvent mixture (diethylether/hexane=70/30 (v/v). The DSC measurement was repeated. The topcurve is the first heat, and the bottom curve is the second heat. Theblock copolymer exhibits a T_(m) between 60 and 70° C. in the first heatindicating presence of a crystalline PEG polymer phase. After cooling,the resulting polymer melts at a rate of 10° C. per minute, the secondheating curve exhibits a low temperature T_(g) indicating a single PEGand PLA mixed phase similar to that observed in FIG. 3 for PLA-PEG blockcopolymer recovered from a polymer melt.

Results shown in FIGS. 3 and 4A suggest that the number and type ofglass transitions observed in block copolymer systems, as well as thevalues of the observed T_(g), vary widely depending upon the sample'shistory. The experimental route to the polymer sample (melt versusprecipitation) alters its phase behavior, thereby leading to distinctglass transition behavior. The thermal history (such as cooling rateafter a melt cycle) also significantly alters phase structure andthermal behavior.

FIG. 4B shows the DSC curves of four PLA-PEG copolymers wherein the PEGblock consists of number average molecular weight of 5 KDa and the PLAblock number average molecular weight is 10, 15, 30 and 50 KDa,respectively. As observed above, heating a polymer sample above itsT_(g) (and/or T_(m) if applicable) erases the effects of thermal historyand allows direct comparison of samples that now have a similar thermalhistory (i.e. have been cooled from a melt at a fixed cooling rate e.g.,10° C./minute). Thus, only second heat data for each system is shown inFIG. 4B and the position of the inflection point (T_(g) value) of themixed PLA and PEG phases increase from −6° C. to 33° C. as PLA numberaverage molecular weight increase from 10 to 50 KDa. FIG. 4C shows thetrend observed within the molecular weight range tested and indicates astrong dependence of T_(g) on polymer molecular weight.

FIG. 5 shows the DSC curve of poly(D,L-lactide) (PLA, Mn=6 kDa) whenrecovered from a precipitation process. The DSC measurement wasrepeated. The top curve is the first heat, and the bottom curve is thesecond heat. FIG. 5 shows a glass transition between 30 and 35° C. inboth first and second heat cycles. As expected for homopolymers, nodifference in the T_(g) is observed despite the different routes(precipitation versus melt) to the polymer sample since no phaseseparation is possible.

FIG. 6A shows the glass transition between 45 and 50° C. observed inpoly(D,L-lactide) (PLA, Mn=75 kDa). The DSC measurement was repeated.The top curve is the first heat, and the bottom curve is the secondheat. The higher T_(g) value of this PLA sample, relative to the lowermolar mass PLA shown in FIG. 5, indicates the expected increase in T_(g)with increasing polymer molecular weight. The first heat cycle in FIG.6A shows an endothermic hysteresis peak on the high temperature side ofthe glass transition indicating that this polymer sample was annealedbelow its T_(g) or cooled (from a melt) at a rate slower than 10° C. perminute (the heating rate used in this heat cycle)

Accurate determination of T_(g) values is difficult when enthalpicrelaxation events occur close to glass transition temperature asobserved for the 75 KDa PLA sample shown in FIG. 6A. FIG. 6B shows thereversing heat component of the total heat flow measured in modulatedDSC experiments for several PLA homopolymers. The number averagemolecular weights of the PLA samples analyzed range between 2K and 120KDa. The data indicates that the T_(g) values increase with molecularweight in the 2 KDa to 22 KDa molecular weight range and then plateauupon further increase in molecular weight as shown in FIG. 6C.

Example 4 Emulsion Preparation of Drug-Containing Nanoparticles

A general emulsion procedure for the preparation of drug loadednanoparticles in aqueous suspension (in 30 wt. % sucrose, 3-6 wt. %polymeric nanoparticles containing about 10 wt. % drug with respect toparticle weight) is summarized as follows. An organic phase is formedcomposed of 30% solids (wt %) including 24% polymer and 6% docetaxel(DTXL). The organic solvents are ethyl acetate (EA) and benzyl alcohol(BA), where BA comprises 21% (wt %) of the organic phase. The organicphase is mixed with an aqueous phase at approximately a 1:2 ratio (oilphase:aqueous phase) where the aqueous phase is composed of 0.5% sodiumcholate, 2% BA, and 4% EA (wt %) in water. The primary emulsion isformed by the combination of the two phases under simple mixing orthrough the use of a rotor stator homogenizer. The primary emulsion isthen formed into a fine emulsion through the use of a high pressurehomogenizer. The fine emulsion is then quenched by addition to a chilledquench (0-5° C.) of deionized water under mixing. The quench:emulsionratio is approximately 10:1. Then, a solution of 35% (wt %) of Tween-80is added to the quench to achieve approximately 4% Tween-80 overall. Thenanoparticles are then isolated and concentrated throughultrafiltration/diafiltration.

In an exemplary procedure to make fast-releasing nanoparticles withsuppressed T_(g), 50% of the polymer is polylactide-poly(ethyleneglycol) diblock copolymer (PLA-PEG; 16 kDa-5 kDa) while 50% of thepolymer is poly(D,L-lactide) (PLA; 6.5 kDa Mn). The resultingnanoparticles have an onset of the glass transition below 37° C., andthus a relatively rapid diffusional-based release at physiologicaltemperature.

In an exemplary procedure to make normal-releasing nanoparticles withaugmented T_(g), 100% of the polymer is polylactide-poly(ethyleneglycol) diblock copolymer (PLA-PEG; 16 kDa-5 kDa). The resultingnanoparticles have an onset of T_(g) at about 37° C.

In an exemplary procedure to make slow-releasing nanoparticles withaugmented T_(g), 50% of the polymer is polylactide-poly(ethylene glycol)diblock copolymer (PLA-PEG; 16 kDa-5 kDa) while 50% of the polymer ispoly(D,L-lactide) (PLA; 75 kDa). The resulting nanoparticles have anonset of T_(g) above 37° C., and thus a relatively slowdiffusional-based release at physiological temperature.

Example 5 Methods for Determining Nanoparticle Size and Drug Content

Particle size was analyzed by two techniques—dynamic light scattering(DLS) and laser diffraction (LD). DLS was performed using a BrookhavenZetaPals instrument at 25° C. in dilute aqueous suspension using a 660nm laser scattered at 90° and analyzed using the Cumulants (typical) andNNLS methods. Laser diffraction was performed with a Horiba LS950instrument in dilute aqueous suspension using both a HeNe laser at 633nm and an LED at 405 nm, scattered at 90°, or an Accusizer SPOS, andanalyzed using the Mie optical model.

Drug load was calculated by dividing the docetaxel content of thenanoparticle slurry by the overall solid content of the slurry.Docetaxel content was determined by extracting the drug from thenanoparticles using acetonitrile and analyzing samples on a C8 reversephase column (Waters X-Bridge C8) using a gradient from 20% acetonitrile(0.016% TFA) to 100% acetonitrile (0.016% TFA). The eluent absorbancewas monitored at 230 nm. Docetaxel eluted with about 40% acetonitrile.The gradient was increased to 100% acetonitrile to elude the hydrophobicnanoparticle polymeric components, PLA-PEG and PLA-PEG-GL2. Thequantitation limit of docetaxel is around 0.5 μg/mL. The solid contentof the slurry is determined gravimetrically by removing water from thesuspension using vacuum and heat.

The particle size and drug loading of the slow, normal, and fastreleasing batches described in Example 4 is summarized in Table 5 below:

TABLE 5 Nanoparticle size and drug content of docetaxel loadednanoparticles Description Diameter via DLS Median via LD Drug load“Fast” Release 120.1 nm Not determined 9.7% “Normal” Release 103.2 nm77.6 nm 9.6% “Slow” Release  99.7 nm 73.8 nm 8.0%

Example 6 Determination of T_(g) for Nanoparticles in Aqueous Suspension

The glass transition temperature (T_(g)) of nanoparticles in aqueoussuspension was determined using heat flux DSC. The samples were heatedbetween 4° C. and 70° C. at a constant heating rate of either 10° C. perminute or 20° C. per minute. Due to the relatively high vapor pressureof water in most of this temperature range, the analysis was conductedin hermetically sealed pans to prevent leakage of water vapor.

FIGS. 8 through 11 show the DSC curves of nanoparticle samples in anaqueous suspension. The nanoparticle samples analyzed were typicallystored frozen at −20° C. The stored samples were first thawed by placingthem into an ambient temperature water bath and subsequentlyconcentrating them using an ultracentrifugation method. About 4 mL ofnanoparticle suspension (30-50 mg/mL nanoparticles) was placed into aMillipore Amicon centrifugal filtration device having polypropylenehousing and a regenerated cellulose membrane filter with a 100 kDamolecular weight cut-off limit. The sample was concentrated bycentrifugation at 4000×g for 1 hour to reduce the retentate volume threeto four fold. The retentate (now about 120-150 mg/mL nanoparticles) wastransferred to a glass scintillation vial and frozen by placing into a−20° C. freezer. This thermal treatment was identical to that used afternanoparticle fabrication and involved temperatures below the glasstransition temperature of the nanoparticles. Thus, this treatment didnot alter the thermal history of the particles relative to theircondition following the nanoparticle fabrication process.

The endothermic transitions shown in FIGS. 8 through 11 were defined byselection of five points to identify the glass transition temperature asshown in FIG. 7. These include a T_(b), the beginning of the deviationof the DSC curve from linearity; T₁, the extrapolated onset temperatureof the glass transition; T₂, the extrapolated end temperature of theglass transition; and T_(e), the end temperature of the glasstransition. The glass transition temperature is defined in one of twopossible ways: as the temperature at half height of the increase in heatcapacity (½ΔC_(p)) (as shown in FIG. 7) or as the point of inflexion.

The DSC curves in FIG. 8 show the endothermic transition exhibited by ananoparticle suspension wherein the particles are made from a 50/50 (byweight) mixture of PLA-PEG (16 kDa-5 kDa) copolymer and low molecularweight PLA homopolymer (Mn=6.5 kDa) when heated between 4° C. and 70° C.at a heating rate of 10° C. per minute. The nanoparticle sample analyzedcontained about 10 wt % encapsulated docetaxel. Analysis was performedusing a TA Instrument Q200 Heat Flux DSC. The DSC measurement wasrepeated. The T_(b) and T_(g) values observed are at 37° C. and 39.7°C., respectively for the first heat cycle (top curve). The sample wassubsequently cooled at rate of 10° C. per minute to 4° C. and heatedagain to 70° C. (at a 10° C. per minute heating rate) in a second heatcycle. The bottom curve of FIG. 8 shows the endothermic transitionobserved in the second heat cycle. The T_(b) and T_(g) values now shiftto 33° C. and 36.7° C., respectively. In use for intravenous injection,nanoparticles would only be exposed to physiological temperature. Thus,only the data in the first heat cycle is of relevance when predictingtheir drug release behavior.

The DSC curves in FIG. 9 show the endothermic transition exhibited by ananoparticle suspension where the particles are made from PLA-PEG (16kDa-5 kDa) copolymer as the only polymeric component in the formulation.The suspension was heated between 4° C. and 70° C. at a heating rate of20° C. per minute. The nanoparticle sample analyzed contained about 10wt % encapsulated docetaxel. Analysis was performed using a TAInstrument Q2000 Heat Flux DSC. The DSC measurement was repeated. TheT_(b) and T_(g) values observed are at 37.3° C. and 41.3° C.,respectively, for the first heat cycle shown as the top curve in FIG. 9.The bottom curve shows a duplicate run of a fresh sample of nanoparticlesuspension, and resulted in T_(b) and T_(g) values of 37.5° C. and 39.8°C., respectively.

The DSC curves in FIG. 10 show the endothermic transition exhibited by ananoparticle suspension wherein the particles are made from a 50/50 (byweight) mixture of PLA-PEG (16 kDa-5 kDa) copolymer and high molecularweight PLA homopolymer (Mn=75 kDa) when heated between 4° C. and 70° C.at a heating rate of 10° C. per minute. The nanoparticle sample analyzedcontained about 10 wt % encapsulated docetaxel. Analysis was performedusing a TA Instrument Q200 Heat Flux DSC. The DSC measurement wasrepeated. The T_(b) and T_(g) values observed are at 43° C. and 44.9°C., respectively, for the first heat cycle (top curve). The sample wassubsequently cooled at rate of 10° C. per minute to 4° C. and heatedagain to 70° C. (at a 10° C. per minute heating rate) in a second heatcycle. The bottom curve shown in FIG. 10 shows the endothermictransition observed in the second heat cycle. The T_(b) and T_(g) valuesshifted to 39° C. and 41° C., respectively.

The DSC curves in FIG. 11A show the endothermic transition exhibited bya nanoparticle suspension wherein the particles are made from a 50/50(by weight) mixture of PLA-PEG (16 kDa-5 kDa) copolymer and highmolecular weight PLA homopolymer (Mn=75 kDa) when heated between 4° C.and 70° C. at a heating rate of 20° C. per minute. The nanoparticlesample analyzed contained about 10 wt % encapsulated docetaxel. Analysiswas performed using a TA Instrument Q2000 Heat Flux DSC. The DSCmeasurement was repeated. The T_(b) and T_(g) values observed are at 44°C. and 45.3° C., respectively, for the first heat cycle. The bottomcurve shows a duplicate run of a fresh sample of nanoparticlesuspension, and gave T_(b) and T_(g) values of 42.4° C. and 43.8° C.,respectively.

Table 6A summarizes the endothermic transitions observed in the DSCstudies on the nanoparticles described above.

TABLE 6A Summary of Endothermic Transitions Observed in the First HeatCycle of Nanoparticle Suspensions Instrument Heating rate FigureDescription used and mode ° C./min. T_(b) (° C.) T_(g) (° C.) T_(e) (°C.) FIG. 8 PLA-PEG and TA Q200-T4 10  35* 39.7 42.5 6.5 kDa PLA FIG. 9PLA-PEG only TA Q2000-T4P 20   37.3 41.3 45.3 FIG. 10 PLA-PEG and TAQ200-T4 10 43 44.9 46.3 75 kDa PLA FIG. 11 PLA-PEG and TAQ2000-T4P 20 4445.3 46.5 75 kDa PLA *In all cases, the T_(b), T_(g), and T_(e) valuescorrespond to the cross-bars on the DSC curves with the exception ofFIG. 8, where the baseline deviation begins at 32° C. Due to anotherstep seen at about 34° C., the cross-bars do not reflect the T_(b) valueaccurately. Thus, a T_(b) value of 35° C. was assigned to account forthe deviation.

To confirm the glass transition temperatures of docetaxel containingnanoparticles, a different batch of particles of the sample compositionas those shown in FIGS. 8 through 11 (and Table 6A) were tested usingmodulated DSC (MDSC). The nanoparticle sample preparation methods usedwere described above to those describe above. The modulated DSCexperiment was conduced using a sinusoidal temperature oscillation ofamplitude 0.5° C. and period of 60 seconds superimposed on a 2°C./minute linear ramp rate. FIG. 11B shows the reversing heat flowcomponent of the total heat flow. These Nanoparticle batches also showeda similar dependence of nanoparticle glass transition temperature on thenanoparticle composition as that seen in FIGS. 8 through 11.

In FIG. 11B, the top M-DSC curve shows a T_(g) at 37.8° C. exhibited bynanoparticles (Sample A) wherein the particles are made from a 50/50 (byweight) mixture of PLA-PEG (16 kDa-5 kDa) copolymer and low molecularweight PLA homopolymer (Mn=6.5 kDa) and contain about 10 wt. %encapsulated docetaxel. The second M-DSC curve shows a T_(g) at 40.1° C.exhibited by nanoparticles (Sample B) where the particles are made fromPLA-PEG (16 kDa-5 kDa) copolymer as the only polymeric component in theformulation and contain about 10 wt. % encapsulated docetaxel. The thirdM-DSC curve shows a T_(g) at 43.0° C. exhibited by nanoparticles (SampleC) where wherein the particles are made from a 50/50 (by weight) mixtureof PLA-PEG (16 kDa-5 kDa) copolymer and high molecular weight PLAhomopolymer (Mn=75 kDa) and contain about 10 wt. % encapsulateddocetaxel. The bottom M-DSC curve shows a T_(g) at 37.3° C. exhibited bynanoparticles (Sample D) where the particles are made from PLA-PEG (16kDa-5 kDa) copolymer as the only polymeric component and contain noencapsulated docetaxel.

Table 6B summarizes the endothermic glass transitions observed in theModulated DSC study of the nanoparticles described above.

TABLE 6B Summary of Glass Transitions observed in Modulated DSC analysisof nanoparticle suspensions Encapsulated Instrument used and T_(g)Figure Description drug mode (° C.) FIG. 11B PLA-PEG and Docetaxel TAQ2000-T4P 37.8 Sample A 6.5 kDa PLA FIG. 11B PLA-PEG only Docetaxel TAQ2000-T4P 40.1 Sample B FIG. 11B PLA-PEG and Docetaxel TA Q2000-T4P 43.0Sample C 75 kDa PLA FIG. 11C PLA-PEG and None TAQ2000-T4P 37.3 Sample D75 kDa PLA

Example 7 Rate of Drug Release from Nanoparticle Samples

The rate of drug released from nanoparticles was determined in vitrounder sink conditions with respect to drug (docetaxel) solubility inwater. The experiment was conducted at physiological pH and ionicstrength using a phosphate buffer saline (PBS) solution containinghydroxypropyl-β-cyclodextrin (HP-βCD) as a drug solubilizer as therelease medium.

Docetaxel loaded nanoparticle samples containing about 5 mg/mL drug werefirst diluted with deionized water to a final concentration of 250 μg/mL(in docetaxel). For example, a batch containing 1 ml of 5 mg/mL DTXL wasdiluted with 19 mL of cold DI-water to give a total of 20 mLnanoparticle suspension containing 250 μg/mL drug.

The release medium (2.5% HP-βCD in PBS (w/w)) was prepared by dissolvingPBS (Sigma PBS P-5368) into deionized water to obtain a phosphate buffersolution containing 0.01M phosphate buffered saline (NaCl: 0.138 M; KCl:0.0027 M). Hydroxypropyl βCD (Trapsol), 100 g, was dissolved into 3900 gof PBS solution to obtain 2.5% HP-βCD in PBS. 120 mL of this releasemedium was transferred into Qorpak wide mouth bottles (16194-290 VWR #,7983 Qorpak #) using a graduated cylinder.

The nanoparticle slurry (3 mL) was added to 120 mL release medium in theQorpak bottle. The bottle was capped and mixed by swirling by hand. Timezero (T=0) control samples were withdrawn. The first non centrifugedsamples were obtained by withdrawing 900 μL of sample immediately afterthe nanoparticle slurry was mixed with the release medium andtransferring it to a HPLC vial containing an equal volume (900 μL) ofacetonitrile. A centrifuged sample (second sample), that underwentcentrifugation treatment analogous to that received by samples drawn atlater time points, was obtained by withdrawing a 4 mL sample from theQorpak bottle and transferring it to a centrifuge tube (Beckman coulter,Capacity 4 mL, #355645). The sample was subsequently centrifuged at50,000 rpm (236,000 g) for 1 hour at 4° C. on an Ultracentrifuge OptimaMAX-XP (P/N 393552AB, TZ08H04, Cat #393315) using a fixed angle rotorMLA 55 (S/N 08U411). 900 μL of supernatant from the upper part of thecentrifuged samples was withdrawn without causing turbulence to avoidre-suspending the nanoparticle pellet at the centrifuge tube bottom.This 900 μL aliquot was transferred to 900 μL of acetonitrile in an HPLCvial. Docetaxel found in the supernatant represents the amount of drugreleased, since any drug still encapsulated within the nanoparticles waspelletized (with the nanoparticles) upon centrifugation.

The release samples in the Qorpac bottles were continually stirred at 75rpm using a waterbath shaker held at 37° C. to maintain continuousmixing conditions. 4 mL samples are drawn at pre-determined time pointsand treated as described above to obtain HPLC samples for analysis ofdocetaxel content. Samples were typically drawn at 0, 1, 2, 4, and 24hours, and the time points were modified as necessary to account forsignificantly faster or slower releasing batches.

FIG. 12 shows the rate of docetaxel released (as percent totalencapsulated docetaxel) at 37±0.5° C. from nanoparticles comprised ofdifferent polymeric materials. The solid line (top) corresponds to drugrelease rate from a “quick” releasing system composed of a 1:1 (w/w)mixture of PLA-PEG (16 kDa-5 kDa) and low molecular weight (Mn=6.5 kDa)PLA homopolymer. The dashed line (middle) corresponds to the drugrelease rate from a “normal or intermediate” releasing system composedof only PLA-PEG (16 kDa-5 kDa). The dotted line (bottom) corresponds todrug release rate from a “slow” releasing system composed of a 1:1 (w/w)mixture of PLA-PEG (16 kDa-5 kDa) and high molecular weight (Mn=75 kDa)PLA homopolymer.

The rate of docetaxel released from the nanoparticles at 37° C. as shownin FIG. 12 indicates a strong dependence on the nanoparticlecomposition. Nanoparticles made from a mixture of PLA-PEG (16 kDa-5 kDa)and PLA (M_(n)=6.5 kDa) exhibited a “quick” release profile with about75% of the encapsulated drug being released within the first 4 hours ofbeing exposed to the release medium. In contrast, when the nanoparticleswere made from only PLA-PEG (16 kDa-5 kDa), about 65% encapsulated drugwas released over a similar period. This effect was further amplifiedwhen the nanoparticles were made from a mixture of PLA-PEG (16 kDa-5kDa) and high molecular weight PLA (M_(n)=75 kDa). In this system, onlyabout 50% of the drug was released in the first 4 hours.

As seen in Table 7A, the endothermic glass transitions observed in thethree nanoparticle systems indicates that the temperature correspondingto the point at which the DSC curve begins to deviate from linearity(T_(b)) is a key parameter in determining the particles' drug releaserate. In the “quick” releasing system, the endothermic transition beginsat 35° C. The observed release behavior in this system indicates thatthe onset of segmental motion and consequent increase in the rate ofdrug diffusion within the nanoparticle core permits drug to reach theparticle-water interface at a rate higher than that observed in systemswhere T_(b)>37° C. The “normal” releasing system exhibits a T_(b)=37.3°C., and the rate of drug release is correspondingly slower. The “slow”releasing system similarly exhibits an even higher T_(b) (43° C.).

The significantly different rates of release observed when T_(b) isbelow, at or above 37° C. is indicative of the strong effect ofsegmental motion on the rate of drug diffusion and release from thenanoparticle. The T_(g) and T_(e) values reported in Table 7 (glasstransition temperature and point at which the DSC curve is linear again)also systematically increase between the “fast,” “normal,” and “slow”releasing systems. This increase is in agreement with the correlationbetween polymer segmental motion and drug release rates and confirmsthat the glass transition temperature of a given nanoparticle systemunder suspension conditions provides a means to predict the relativerate of drug release. The absolute rate of release additionally dependson other factors including the polymer-drug miscibility, drug solubilityin water, drug molecular size, and drug phase structure (amorphous orcrystalline) within the nanoparticle.

The reversing heat component of the total heat flow observed in MDSCexperiments is free from artifacts resulting from enthalpic relaxationsoccurring close to the endothermic glass transition. As such, MDSCprovides a more accurate determination of the T_(g) value when comparedto conventional DSC. MDSC analysis (data shown in Table 7B) illustratesthat nanoparticles made from a mixture of PLA-PEG (16 kDa-5 kDa) and PLA(M_(n)=6.5 kDa) that exhibit a Tg at 37.8° C. also exhibit a “quick”release profile with about 75% of the encapsulated drug being releasedwithin the first 4 hours of being exposed to the release medium. Thisindicates that when nanoparticle glass transition temperatures are closeto 37° C. (physiological temperature), high degree of segmental motionleads to relatively fast release of encapsulated drug. When thenanoparticles composed of PLA-PEG (16 kDa-5 kDa) only, about 65%encapsulated drug was released over a similar period of time. Lowerdegree of segmental motion in this higher T_(g) (40.1° C.) sampleresults in correspondingly slower release of encapsulated drug. Thistrend continues in nanoparticles made from a mixture of PLA-PEG (16kDa-5 kDa) and high molecular weight PLA (M_(n)=75 kDa), where a evenhigher T_(g) (=43.0° C.) leads to a correspondingly slower release ofencapsulated drug with only about 50% of the drug being released in thefirst 4 hours. Entry 4 (Table 7B) shows that the glass transitiontemperature of a polymeric nanoparticle composed of PLA-PEG (16 kDa-5kDa) with no encapsulated drug. Comparison with a drug containingnanoparticle of similar polymer composition (entry 1, Table 7B) thatcontains encapsulated drug illustrates that docetaxel measurably impactsthe glass transition temperature of a polymeric nanoparticle. In thiscase, about 10 wt. % encapsulated docetaxel increases the Tg value by0.5° C. This body of data indicates that T_(g) values of drug containingnanoparticles provide a means to accurately predict the correspondingdrug release rates. Other measurements such as glass transitiontemperature of the polymeric components and/or their physical mixtureswith drugs can provide general trends, for instance the effect of changein polymer molecular weight. However, such measurements fail to enableclassification of a specific drug bearing nanoparticle as fast, moderateor slow releasing

DSC data shown in FIGS. 3 through 7 for PLA-PEG recovered from a melt orprecipitation and low (M_(n)=6.5 kDa) or high (M_(n)=75 kDa) molar massPLA illustrates the strong dependence of the thermal behavior of thesepolymeric nanoparticle components on their thermal history as well as onthe route of polymer processing prior to analysis. Thus, the glasstransitions observed for polymeric components do not directly correlateto the thermal characteristics of nanoparticles. The process ofnanoparticle fabrication imparts a specific thermal history to componentpolymers and additionally imparts specific morphological and phasecharacteristics to block copolymers such as PLA-PEG.

The thermal behavior of the polymer components affects the glasstransition temperature in a predictable manner. For example,nanoparticles made from a 1:1 (w/w) mixture of low molecular weight PLA(M_(n)=6.5 kDa) and PLA-PEG (16 kDa-5 kDa) exhibit a lower T_(g) thanthat exhibited by nanoparticles made from a similar mixture except withhigh molecular weight PLA (75 kDa). This result is based upon T_(g) (75kDa PLA) being greater than T_(g) (6.5 kDa PLA) by about 20° C. Thedifference in T_(g) permits selection of nanoparticle compositions tomodulate the rate of drug release. However, the actual transitiontemperatures of nanoparticles made form such polymeric components cannotbe predicted from T_(g) values of polymeric components.

Example 8 Effect of Temperature on the Rate of Drug Release

To further test the role of nanoparticle glass transition temperature inthe “fast,” “normal,” and “slow” releasing systems, the rates of drugrelease were tested at three temperatures other than 37° C. Thesetemperatures included 32° C., i.e., 3-5° C. below the lowest onsettemperature (T_(b)), and 52° C., a similar increment above the highesttransition end temperature (T_(e)). In addition, the rate of release wasdetermined at 25° C. to check the behavior at a temperature considerablybelow the T_(g)'s of all three nanoparticle systems.

The results are shown in FIG. 13, with an expansion of the 1-4 hour timeperiod shown in FIG. 14. At 25° C., all three nanoparticle systems,despite their different onset of glass transition (T_(b)) temperatures,exhibited a similar rate of drug release (see data points shown astriangles in FIGS. 13 and 14). This observation confirms that attemperatures well below the T_(g)'s of all three systems, segmentalmotion is limited in all cases such that rates of drug transport andrelease are similar and relatively slow.

At 52° C., all three nanoparticle systems exhibit an accelerated rate ofdrug release confirming that at temperatures well above their glasstransition temperatures, the nanoparticle cores are equally plastic(rubbery). Thus, drug diffusion rates are comparable and release ratesbecome nearly identical with almost all drug being released within thefirst 30 minutes at 52° C. (see data shown as squares in FIGS. 13 and14). The burst release observed for the three nanoparticle systems at52° C. mirrors the trends observed at 37° C., with “slow”, “normal” and“fast” systems releasing 45%, 65%, and 75% of their drug contentimmediately.

At 32° C., the release rates are similar but higher (for all systems)than those observed at 25° C. (see data shown as circles in FIGS. 13 and14). These results suggest a temperature dependence of drug diffusioncoefficient in the polymeric core and water solubility, with bothparameters increasing at higher temperature and leading to the observedincrease in drug release rates.

Example 9 Docetaxel Nanoparticles

Docetaxel nanoparticles comprising various PLA-PEG copolymers areprepared using the following formulation: 10% (w/w) theoretical drug and90% (w/w) polymer-PEG (16-5, 30-5, 50-5, or 80-5 PLA-PEG). % totalsolids=20%. Solvents used are 21% benzyl alcohol and 79% ethyl acetate(w/w). For a 1 gram batch size, 100 mg of drug is mixed with 900 mg ofpolymer-PEG (16-5, 30-5, 50-5, or 80-5 PLA-PEG).

Docetaxel nanoparticles are produced as follows. In order to prepare adrug/polymer solution, appropriate amounts of docetaxel, and polymer areadded to a glass vial along with appropriate amounts of ethyl acetateand benzyl alcohol. The mixture is vortexed until the drug and polymerare completely dissolved.

An aqueous solution is prepared. The aqueous phase for the 16-5 PLA-PEGformulation contains 0.5% sodium cholate, 2% benzyl alcohol, and 4%ethyl acetate in water. The 30-5 PLA-PEG formulation contains 5% sodiumcholate, 2% benzyl alcohol, and 4% ethyl acetate in water, % totalsolids=20%. The aqueous phase for the 50-5 PLA-PEG formulation contains5% sodium cholate, 2% benzyl alcohol, and 4% ethyl acetate in water, %total solids=20%. The aqueous phase for the 80-5 PLA-PEG formulationcontains 5% sodium cholate, 2% benzyl alcohol, and 4% ethyl acetate inwater, % total solids=20%. When higher molecular weight polymer-PEG isused (i.e. 30-5, 50-5, or 80-5 PLA-PEG), the concentration of sodiumcholate surfactant in the water phase is increased from 0.5% to 5% inorder to obtain nanoparticles with sizes similar to those particlescomprising 16-5 PLA-PEG. Specifically, appropriate amounts of sodiumcholate and DI water are added to a bottle and mixed using a stir plateuntil they are dissolved. Subsequently, appropriate amounts of benzylalcohol and ethyl acetate are added to the sodium cholate/water mixtureand mixed using a stir plate until all are dissolved.

An emulsion is formed by combining the organic phase into the aqueoussolution at a ratio of 5:1 (aqueous phase:oil phase). The organic phaseis poured into the aqueous solution and homogenized using handhomogenizer at room temperature to form a coarse emulsion. The solutionis subsequently fed through a high pressure homogenizer (110S) to form ananoemulsion.

The emulsion is quenched into cold DI water at <5° C. while stirring ona stir plate. The ratio of Quench to Emulsion is 8:1. Tween 80 in wateris then added to the quenched emulsion at a ratio of 25:1 (Tween80:drug).

The nanoparticles are concentrated through tangential flow filtration(TFF) followed by diafiltration to remove solvents, unencapsulated drugand Tween 80 (solubilizer). A quenched emulsion is initiallyconcentrated through TFF using a 300 KDa Pall cassette (Two 0.1 m²membranes) to an approximately 100 mL volume. This is followed bydiafiltration using approximately 20 diavolumes (2 L) of cold DI water.The volume is minimized before collecting, then 100 mL of cold water isadded to the vessel and pumped through the membrane for rinsing.Approximately 100-180 mL of material in total are collected in a glassvial. The nanoparticles are further concentrated using a smaller TFF toa final volume of approximately 10-20 mL.

In order to determine the solids concentration of unfiltered finalslurry, a volume of final slurry is added to a tared 20 mL scintillationvial and dried under vacuum on lyo/oven. Subsequently the weight ofnanoparticles is determined in the volume of the dried down slurry.Concentrated sucrose (0.666 g sucrose/g total) is added to the finalslurry sample to attain a final concentration of 10% sucrose.

In order to determine the solids concentration of a 0.45 μm filteredfinal slurry, a pre-determined volume of the final slurry sample isfiltered before the addition of sucrose using a 0.45 μm syringe filter.A volume of the filtered sample is then added to a tared 20 mLscintillation vial, dried under vacuum on lyo/oven, and the weightdetermined gravimetrically. The remaining sample of unfiltered finalslurry is frozen with sucrose.

Table A provides the particle size and drug load of the docetaxelnanoparticles produced as described above.

TABLE A Polymer Load DTXL % Size (nm) 16/5 PLA/PEG 4.05 110.70 30/5PLA/PEG 1.48 129.00 50/5 PLA/PEG 2.75 170.60 80/5 PLA/PEG 3.83 232.00

As shown in Table A, docetaxel nanoparticles comprising 50-5 PLA-PEG and80-5 PLA-PEG result in a drug load of about 2.75% and 3.83%,respectively.

In vitro release test is performed on the above described docetaxelnanoparticles. As depicted in FIG. 15, nanoparticles fabricated using50-5 PLA-PEG or 80-5 PLA-PEG slowed down the release of docetaxel fromthe nanoparticles compared with nanoparticles having lower molecularPLA-PEG.

Example 10 Bortezomib Nanoparticles

Bortezomib nanoparticles comprising various PLA-PEG copolymers areprepared using the following formulation: 30% (w/w) theoretical drug and70% (w/w) polymer-PEG (16/5, 30-5, 50-5, 65-5, or 80-5 PLA-PEG). % Totalsolids=20%. Solvents used are 21% benzyl alcohol and 79% ethyl acetate(w/w). For a 1 gram batch size, 300 mg of drug is mixed with 700 mg ofpolymer-PEG (16/5, 30-5, 50-5, 65-5, or 80-5 PLA-PEG).

Bortezomib nanoparticles are prepared using a protocol similar to theprotocol described above for docetaxel nanoparticles.

Table B provides the particle size and drug load of the bortezomibnanoparticles produced as described above.

TABLE B Polymer Load BTZ % Size (nm) 16/5 PLA/PEG 3 107 30/5 PLA/PEG 1108 50/5 PLA/PEG 2.7 106 65/5 PLA/PEG 0.2 155 80/5 PLA/PEG 0.67 149

In vitro release test is performed on the above described bortezomibnanoparticles. As depicted in FIG. 16, incorporation of 50-5 PLA-PEGslowed down the release of bortezomib from the nanoparticles.

Example 11 Vinorelbine Nanoparticles

Vinorelbine nanoparticles comprising either 16-5 or 50-5 PLA-PEGcopolymer are prepared using the following formulation: 20% (w/w)theoretical drug and 80% (w/w) polymer-PEG (16/5 or 50-5 PLA-PEG). Fornanoparticles comprising 16-5 PLA-PEG: % Total solids=20%; fornanoparticles comprising 50-5 PLA-PEG: % Total solids=30%. For allnanoparticles: solvents used are 21% benzyl alcohol and 79% ethylacetate (w/w).

Vinorelbine nanoparticles are prepared using a protocol similar to theprotocol described above for docetaxel nanoparticles.

Table C provides the particle size and drug load of the vinorelbinenanoparticles produced as described above.

TABLE C Polymer Drug Load % Size (nm) 16/5 PLA/PEG 10 101 50/5 PLA/PEG8.4 109

In vitro release test is performed on the above described vinorelbinenanoparticles. As depicted in FIG. 17, nanoparticles made using 50-5PLA-PEG resulted in slower release of vinorelbine from the nanoparticlescompared to nanoparticles made with 16-5 PLA-PEG.

Example 12 Vincristine Nanoparticles

Vincristine nanoparticles comprising either 16-5 or 50-5 PLA-PEGcopolymer are prepared using the following formulation: 20% (w/w)theoretical drug and 80% (w/w) polymer-PEG (16/5 or 50-5 PLA-PEG). Fornanoparticles comprising 16-5 PLA-PEG: % Total solids=40%; fornanoparticles comprising 50-5 PLA-PEG: % Total solids=20%. For allnanoparticles: solvents used are 21% benzyl alcohol and 79% ethylacetate (w/w).

Vincristine nanoparticles are prepared using a protocol similar to theprotocol described above for docetaxel nanoparticles.

Table D provides the particle size and drug load of the vinorelbinenanoparticles produced as described above.

TABLE D Polymer Drug Load % Size (nm) 16/5 PLA/PEG 2.9 103 50/5 PLA/PEG2.8 122

In vitro release test is performed on the above described vincristinenanoparticles. As depicted in FIG. 18, incorporation of 50-5 PLA-PEGslowed down the release of vincristine from the nanoparticles.

Example 13 Bendamustine Nanoparticles

Bendamustine HCl nanoparticles comprising either 16-5 or 50-5 PLA-PEGcopolymer are prepared using the following formulation: 17% (w/w)theoretical drug and 83% (w/w) polymer-PEG (16/5 or 50-5 PLA-PEG) at 20%(w/w) polymer concentration in methylene chloride. Bendamustine HCl iscomplexed with sodium tetraphenylborate at a 1:1 ratio. % Totalsolids=40%. Solvents used are 32% benzyl alcohol and 68% methylenechloride (w/w).

Bendamustine nanoparticles are prepared using a protocol similar to theprotocol described above for docetaxel nanoparticles, with an additionalstep in which the methylene chloride is removed from the emulsion on arotovapor by pulling vacuum while the emulsion is rotating in an icebath for 10 minutes.

Table E provides the particle size and drug load of the vinorelbinenanoparticles produced as described above.

TABLE E Polymer Drug Load % Size (nm) 16/5 PLA/PEG 3.5 97 50/5 PLA/PEG2.1 202

In vitro release test is performed on the above described bendamustinenanoparticles. As depicted in FIG. 19, nanoparticles made using 50-5PLA-PEG demonstrated slower release of bendamustine from thenanoparticles compared to nanoparticles made using 16-5 PLA-PEG.

Example 14 Nanoparticle Preparation with Epothilone

Epothilone B nanoparticles were produced using the followingformulations:

10% (w/w) theoretical drug

90% (w/w) Polymer-PEG, 16-5 PLA-PEG or 50-5 PLA-PEG

% Total Solids=20%

Solvents: 21% benzyl alcohol, 79% ethyl acetate (w/w)

For a 1 gram batch size, 100 mg of drug was mixed with 900 mg ofPolymer-PEG: 16-5 or 50-5 PLA-PEG.

Epothilone B nanoparticles were produced as follows. In order to preparea drug/polymer solution, 100 mg of epothilone B was added to a 7 mLglass vial along with 3.16 g of ethyl acetate. The mixture was vortexeduntil the drug was mostly dissolved. Subsequently, 0.840 g of benzylalcohol was added to the glass vial and vortexed until the drug wascompletely dissolved. Lastly, 900 mg of polymer-PEG was added to themixture and vortexed until everything was dissolved.

An aqueous solution for either a 16-5 PLA-PEG formulation or a 50-5PLA-PEG formulation was prepared. The aqueous solution of the 16-5PLA-PEG formulation contained 0.1% Sodium Cholate, 2% Benzyl Alcohol,and 4% Ethyl acetate in water. Specifically, 1 g of sodium cholate and939 g of DI water were added to a 1 L bottle and mixed using a stirplate until they were dissolved. Subsequently, 20 g of benzyl alcoholand 40 g of ethyl acetate were added to the sodium cholate/water mixtureand mixed using a stir plate until all were dissolved. The aqueoussolution for the 50-5 PLA-PEG formulation contained 5% Sodium Cholate,2% Benzyl Alcohol, and 4% Ethyl acetate in water. Specifically, 50 gsodium cholate and 890 g of DI water were added to a 1 L bottle andmixed using a stir plate until they were dissolved. Subsequently, 20 gof benzyl alcohol and 40 g of ethyl acetate were added to the sodiumcholate/water mixture and mixed using a stir plate until all weredissolved.

An emulsion was formed by combining the organic phase into the aqueoussolution at a ratio of 5:1 (aqueous phase:oil phase). The organic phasewas poured into the aqueous solution and homogenized using a handheldhomogenizer for 10 seconds at room temperature to form a coarseemulsion. The solution was subsequently fed through a high pressurehomogenizer (110S). For the 16-5 PLA-PEG formulation, the pressure wasset to 9000 psi for two discreet passes to form the nanoemulsion. Forthe 50-5 PLA-PEG formulation, the pressure was set to 45 psi on gauge(9900 psi) for two discreet passes and then increased to 60 psi on gauge(13200 psi) for two additional passes.

The emulsion was quenched into cold DI water at <5° C. while stirring ona stir plate. The ratio of Quench to Emulsion was 8:1.35% (w/w) Tween 80in water was then added to the quenched emulsion at a ratio of 25:1(Tween 80:drug).

The nanoparticles were concentrated through tangential flow filtration(TFF) followed by diafiltration to remove solvents, unencapsulated drugand solubilizer. A quenched emulsion was initially concentrated throughTFF using a 300 KDa Pall cassette (Two 0.1 m² membranes) to anapproximately 100 mL volume. This was followed by diafiltration usingapproximately 20 diavolumes (2 L) of cold DI water. The volume wasminimized before collection, then 100 mL of cold water was added to thevessel and pumping through the membrane for rinsing. Approximately100-180 mL of material was collected in a glass vial. The nanoparticleswere further concentrated using a smaller TFF to a final volume ofapproximately 10-20 mL.

In order to determine the solids concentration of unfiltered finalslurry, a volume of final slurry was added to a tared 20 mLscintillation vial and dried under vacuum on lyo/oven. Subsequently theweight of nanoparticles was determined in the volume of the dried downslurry. Concentrated sucrose (0.666 g sucrose/g total) was added to thefinal slurry sample to attain a final concentration of 10% sucrose.

In order to determine the solids concentration of 0.45 μm filtered finalslurry, a portion of the final slurry sample was filtered before theaddition of sucrose using a 0.45 μm syringe filter. A volume of thefiltered sample was then added to a tared 20 mL scintillation vial anddried under vacuum on lyo/oven. The remaining sample of unfiltered finalslurry was frozen after dissolving sucrose (10 wt) in it.

Particle size was analyzed by two techniques—dynamic light scattering(DLS) and laser diffraction. DLS was performed using a BrookhavenZetaPals instrument at 25° C. in dilute aqueous suspension using a 660nm laser scattered at 90° and analyzed using the Cumulants and NNLSmethods (TP008). Laser diffraction was performed with a Horiba LS950instrument in dilute aqueous suspension using both a HeNe laser at 633nm and an LED at 405 nm, scattered at 90° and analyzed using the Mieoptical model (TP009).

Table F gives the particle size and drug load of the particles describedabove.

TABLE F EpoB Particle Load Size Formulation Description (%) (nm) 16/5PLA/PEG 20% solids, 2 passes at 9900 psi 2.3 91 50/5 PLA/PEG 20% solids,2 passes at 9900 psi and 2 1.6 174 passes at 13200 psi

Example 15 In Vitro Release

To determine the in vitro release of epothilone B from thenanoparticles, the nanoparticles were suspended in PBS release media andincubated in a water bath at 37° C. Samples were collected at specifictime points. An ultracentrifugation method was used to separate releaseddrug from the nanoparticles.

FIG. 20 shows the results of an in vitro release study on the 16-5PLA-PEG and 50/5 PLA/PEG formulations. Data shows 100% release of Epo Bfrom the 16/5 PLA/PEG formulation after one hour. The 50/5 PLA/PEGformulation is a slower releasing formulation with 50% release at 1hour, 60% release at 2 hours, 70% release at 4 hours, and greater than80% drug release at 24 hours. The two formulations demonstrate theability to encapsulate epothilone B into nanoparticles and the abilityto impact in vitro release through the selection of the polymer typeused in the formulation.

Example 16 Nanoparticle Preparation—Budesonide

All budesonide batches were produced as follows, unless noted otherwise.Drug and polymer (16/5 PLA-PEG) constituents were dissolved in the oilphase organic solvent system, typically 70% ethyl acetate (EA) and 30%benzyl alcohol (BA), at 20% or 30 wt % [solids]. The aqueous phaseconsisted mainly of water, pre-saturated with 2% benzyl alcohol and 4%ethyl acetate, with sodium cholate (SC) as surfactant. The coarse 0/Wemulsion was prepared by dumping the oil phase into the aqueous phaseunder rotor stator homogenization at an oil:aqueous ratio of 1:5 or1:10. The fine emulsion was then prepared by processing the coarseemulsion through a Microfluidics high pressure homogenizer (generallyM110S pneumatic) at 9000 psi through a 100 μm Z-interaction chamber. Theemulsion was then quenched into a cold DI water quench at 10:1 or 5:1quench:emulsion ratio. Polysorbate 80 (Tween 80) was then added as aprocess solubilizer to solubilize the unencapsulated drug. The batch wasthen processed with ultrafiltration followed by diafiltration to removesolvents, unencapsulated drug and solubilizer. This process is depictedpictorially in FIGS. 1 and 2.

The particle size measurements were performed by Brookhaven DLS and/orHoriba laser diffraction. To determine drug load, slurry samples weresubmitted for HPLC and [solids] analysis. The slurry retains were thendiluted with sucrose to 10% before freezing. All ratios listed are on aw/w basis, unless specified otherwise. Tween 80 may be used post quenchto remove unencapsulated drug.

An in vitro release method is used to determine the initial burst phaserelease from nanoparticles at both ambient and 37° C. conditions.Nanoparticles are placed into sink conditions for the API and mixed in awater bath. Released and encapsulated drug are separated by using anultracentrifuge.

The centrifugal system is run as follows: 3 mL slurry of budesonidenanoparticles (approx 250 μg/mL budesonide PLGA/PLA nanoparticles) inDI-water is placed into glass bottles containing 130 ml release media(2.5% hydroxyl beta cyclodextrin in 1×PBS), which is continually stirredat 150 rpm using a shaker. At pre-determined time points, aliquot ofsamples (4 mL) were withdrawn. Samples are centrifuged at 236,000 g for60 minutes and the supernatant is assayed for budesonide content tomeasured released budesonide.

Particle size is analyzed by two techniques—dynamic light scattering(DLS) and laser diffraction. DLS is performed using a BrookhavenZetaPals instrument at 25° C. in dilute aqueous suspension using a 660nm laser scattered at 90° and analyzed using the Cumulants and NNLSmethods. Laser diffraction is performed with a Horiba LS950 instrumentin dilute aqueous suspension using both a HeNe laser at 633 nm and anLED at 405 nm, scattered at 90° and analyzed using the Mie optical model(TP009).

Example 17

Nanoparticles with various drug loads were prepared by varying thefollowing parameters: vary Q:E ratio (5:1, 15:1 and 30:1); increase[solids] to 30% by reducing initial [budesonide] to 10%; increaseparticle size by reducing surfactant to 0.5%.

A single emulsion was made at 30% solids, 10% drug, and emulsion wassplit into three different quenches at Q:E ratios of 5:1, 15:1 and 30:1.The particle size was 137 nm and drug load ranged from 3.4% to 4%. Theincreased drug load may be due to increased [solids] and particle size,while varying Q:E ratio did not seem to have a significant effect ondrug load.

A 10 g batch was made for scale up using the formulation and process ofExample 16, using 30% solids and 10% Microfluidics M110EH electric highpressure homogenizer was used to make this batch at 900 psi using a 200μm Z-chamber. Particle size was 113 nm and drug load was 3.8% (Batch55-40, control).

Example 18 Nanoparticles

Various batches on nanoparticles were prepared using the generalprocedure of Example 16, and using the following parameters

16/5 PLA-PEG with mid MW PLA (IV (inherent viscosity)=0.3) at 40%solids: Batch #52-198

16/5 PLA-PEG with 40%[solids]; 10% drug, using 60/40 of ethylacetate/benzyl alcohol Batch: #58-27-1

16/5 PLA-PEG with 40% [solids] and 5% [drug]: Batch #58-27-2

16/5 PLA-PEG with high MW PLA (IV=0.6-0.8) at 40% solids: Batch#41-171-A

High MW PLA (IV=0.6-0.8) with DSPE-PEG (2 k): Batch #41-171-B & 61-8-B

16/5 PLA-PEG with high MW PLA (IV=0.6-0.8) at 75% solids: Batch #41-176

16/5 PLA-PEG with doped high MW PLA (IV=0.6-0.8) at 75% and 50% solids:Batch #41-183-A&B

Mid MW PLA was obtained from Surmodics (aka Lakeshore (LS)), with aninherent viscosity of 0.3. 16/5 PLA-PEG was obtained from BoehringerIngelheim (batch 41-176) or Polymer Source (batch 41-183). High MW PLAwith a M_(n) of 80 kDa, M_(w) of 124 kDa was obtained from Surmodics.

Table G indicates the size and drug load of the nanoparticle batches:

Size Drug Batch No# Description (nm) Load 52-198 Doped mid MW PLA 1207.08% 58-27-1 Higher [solids] 153 4.28% 58-27-2 Higher [solids] andlower initial [drug] 101 1.92% 41-171-A Doped high MW PLA 117 4.21%41-171-B High MW PLA with DSPE-PEG 224 6.11% 41-176 Doped high MW PLA @75% 181 5.14% 41-183-A Doped high MW PLA @ 75% 176 1.90% 41-183-B Dopedhigh MW PLA @ 50% 125 1.72% 61-8-B High MW PLA with DSPE-PEG 168  3.2%In vitro release of each batch is depicted in FIG. 21. Note: Batch41-171-A at 1 hour time point is an outlier caused by one of theuncentrifuged samples reading extraordinarily low. Both batches 41-171-B(lipid formulation) and 41-183-A (high MW PLA) showed drug release ≦50%at 2 hours while the other formulations had released between 70-100%within 2 hours.

Example 19 Batch for Animal study

A 10 g batch was made to confirm the drug load and release seen inbatches 41-176 and 41-183-A as well as to provide material for animalstudies. Particle size was 183 nm and drug load was 5.03%. Formulationand process parameters were scaled linearly with the exception of waterphase [surfactant]. Table H below details the major differences betweenthe batches:

TABLE H Parameter or attribute 41-176 41-183A 62-30 Scale 1 g 1 g 10 g16/5 Polymer supply Boehringer Polymer Boehringer Ingelheim IngelheimSource Sodium cholate   2%   5%  2.5% Homogenizer M110S M110S M110EHDrug load 5.14% 1.73% 5.03% PSD 181 176 183

The 10 g batch, batch no. 62-30, was chosen for the PK study and wasfirst tested for drug release to ensure the release was similar to41-176 and 41-183-A, as shown in FIG. 22.

Example 20 Rat Study: Pharmacokinetics

Rats (male Sprague Dawley, approximately 300 g with jugular cannulae)were given a single intravenous dose of 0.5 mg/kg of budesonide orpassively targeted nanoparticles (PTNP) encapsulating budesonide(prepared as in Example 16) at time=0. At various times after dosing,blood samples were collected from the jugular cannulae into tubescontaining lithium heparin, and plasma was prepared. Plasma levels weredetermined by extraction of the budesonide from plasma followed by LCMSanalysis. The results from this PK study are shown in FIG. 23.

Encapsulation of budesonide in co-polymer nanoparticles resulted in an11-fold increase in the maximum plasma concentration (Cmax), a 4-foldincrease in half-life (t_(1/2)) and a 36-fold increase in the area underthe concentration—time curve (AUC). Budesonide encapsulation alsoreduces the volume of distribution (Vz) by 9-fold and reduces theclearance from plasma (Cl) by 37-fold. Each of these parameter changesindicates that nanoparticle encapsulation of budesonide promotes plasmalocalization of budesonide at the expense of tissue distribution of thesteroid. Table I outlines the pharmacokinetic analyses of budesonide andbudesonide PTNP.

TABLE I Group 1: Budesonide Rat 1-1 Rat 1-2 Rat 1-3 Rat 1-4 Rat 1-5 Rat1-6 Avg sd Cmax (ng/mL) 324 241 226 337 306 279 286 45 t_(1/2) (hr) 0.730.75 0.74 0.69 0.70 0.87 0.75 0.06 AUC_(inf) (hr*ng/mL) 159 126 108 161159 173 148 25 Vz (mL/kg) 3311 4291 4937 3104 3191 3627 3744 727 Cl(mL/hr/kg) 3136 3966 4611 3104 3151 2892 3477 668 Group 2: BudesonidePTNP Rat 2-1 Rat 2-2 Rat 2-3 Rat 2-4 Rat 2-5 Rat 2-6 Avg sd Cmax (ng/mL)3180 3750 4140 3330 2800 2260 3243 669 t_(1/2) (hr) 2.66 2.95 3.18 3.002.98 2.8 2.9 0.2 AUC_(inf) (hr*ng/mL) 5614 6666 5760 4686 4299 5245 5378839 Vz (mL/kg) 341 319 398 463 501 383 400.8 70.0 Cl (mL/hr/kg) 89 75 87107 116 95 94.8 14.7

Example 21 Rat Model of Inflammatory Disease

Budesonide and Budesonide PTNP were compared in a model of inflammatorybowel disease (IBD) as an efficacy model of inflammation. In this model,female rats were given two subcutaneous doses of 8 mg/kg indomethacin at24 hour intervals to induce lesions resembling those occurring inCrohn's disease in the small intestine. Intravenous daily treatment withvehicle, budesonide (0.02, 0.2 or 2 mg/kg) or budesonide PTNP (0.02, 0.2or 2 mg/kg) or oral daily treatment with Dexamethasone (0.1 mg/kg) wasinitiated one day before indomethacin treatment (day −1) and continuedfor 5 total days (days −1 to 3). Animals were euthanized on day 4, and a10 cm area at risk in the small intestine was scored for gross pathologyand weighed.

Using a disease scoring system in which a score of 0 is normal, and ascore of 5 indicates death due to IBD symptoms, normal rats have anaverage score of 0, and an average intestinal weight of 0.488 g. Incontrast, vehicle treated controls with indomethacin-induced IBD had anaverage clinical score of 2.7 (FIG. 24) and intestinal weight of 2.702 g(FIG. 25). Intestinal scores were significantly decreased towards normalafter treatment with budesonide at doses of 0.02 mg/kg (52% decrease),0.2 mg/kg (53% decrease) and 2 mg/kg (59% decrease; FIG. 24). In thesame way, intestinal scores were significantly decreased towards normalafter treatment with budesonide PTNP at doses of 0.02 mg/kg (59%decrease), 0.2 mg/kg (96% decrease) and 2 mg/kg (93% decrease; FIG. 24).Small intestine scores were also significantly decreased by treatmentwith 0.2 mg/kg budesonide PTNP (94%) or 2 mg/kg budesonide PTNP (85%)compared to animals treated with the same dose of budesonide free-drug(FIG. 25).

Small intestine weights were significantly decreased toward normalfollowing treatment with budesonide at doses of 0.02 mg/kg (52%decrease), 0.2 mg/kg (53% decrease) or 2 mg/kg (59% decrease; FIG. 8).In the same way, intestinal weights were significantly decreased towardsnormal after treatment with budesonide PTNP at doses of 0.02 mg/kg (64%decrease), 0.2 mg/kg (93% decrease) and 2 mg/kg (90% decrease; FIG. 25).Small intestine weights were also significantly decreased by treatmentwith 0.2 mg/kg budesonide PTNP (86%) or 2 mg/kg budesonide PTNP (74%)compared to animals treated with the same dose of budesonide free-drug(FIG. 24). Results of this study indicate that daily intravenoustreatment with budesonide or budesonide PTNP significantly inhibited theclinical parameters associated with indomethacin induced inflammatorybowel disease in rats, with budesonide PTNP treatment having asignificant beneficial effect over treatment with budesonide atcorresponding dose levels.

Example 22 Particles with Alternate Co-Polymers

Following the general procedure of Example 16, nanoparticles were formedfrom budesonide with PLA-PEG co-polymers as follows:

50/5 PLA-PEG: (PLA Mw=50; PEG Mw=5); Batch #55-106-A

50/5 PLA-PEG and high MW (75 Mn PLA: Batch #55-106-B

80/10 PLA-PEG: Batch #55-106-A

80/10 PLA-PEG and high MW PLA: 55-106-B

Batches B and D had high MW 75 Mn PLA doped at 50% of total polymer.Table J shows drug loading weight percent:

Batch # Description Drug Load 55-106-A 50/5 PLA-PEG 2.30% 55-106-B 50/5PLA-PEG with high MW PLA doped 3.10% 55-106-C 80/10 PLA-PEG 1.40%55-106-D 80/10 PLA-PEG with high MW PLA doped 1.50%

A drug release study was performed to see whether changing copolymer MWhad an effect on slowing down drug release. FIG. 26 shows batch#55-106-B, i.e. 50/5 PLA-PEG doped with high MW PLA, appears similar tothe control formulation, 62-30.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

INCORPORATION BY REFERENCE

The entire contents of all patents, published patent applications,websites, and other references cited herein are hereby expresslyincorporated herein in their entireties by reference.

The invention claimed is:
 1. A method of treating asthma,osteoarthritis, dermatitis, inflammatory bowel disease, ulcerativecolitis, or Crohn's disease, comprising administering to a patient inneed thereof an effective amount of a pharmaceutical aqueous suspensioncomprising a plurality of moderate release biocompatible, therapeuticnanoparticles having a glass transition temperature between about 39° C.and about 41° C., wherein the nanoparticles comprise about 3 to about 40weight percent of budesonide, and poly(lactic)acid-poly(ethylene)glycol,wherein the therapeutic nanoparticles have a diameter of about 80 nm toabout 130 nm.
 2. The method of claim 1, wherein the poly(ethylene)glycolportion of the copolymer has a number average molecular weight of about4 kDa to about 6 kDa.
 3. The method of claim 1, wherein thepoly(lactic)acid portion of the copolymer has a number average molecularweight of about 15 kDa to about 20 kDa.
 4. The method of claim 1,wherein the poly(lactic)acid portion of the copolymer has a numberaverage molecular weight of about 16 kDa, and the poly(ethylene)glycolportion of the copolymer has a number average molecular weight of about5 kDa.
 5. The method of claim 1, comprising about 50 to about 90% byweight of the poly(lactic)acid-poly(ethylene)glycol copolymer.
 6. Themethod of claim 1, wherein the patient is suffering from inflammatorybowel disease.
 7. The method of claim 1, wherein the nanoparticlescomprise about 4 to about 25 weight percent of the budesonide.
 8. Themethod of claim 1, wherein the nanoparticles comprise about 5 to about30 weight percent of the budesonide.
 9. A method of treatinginflammatory bowel disease, comprising administering to a patient inneed thereof an effective amount of a pharmaceutical suspensioncomprising a plurality of biocompatible, therapeutic nanoparticleshaving a glass transition temperature between about 39° C. and about 41°C. and a diameter of about 80 nm to about 130 nm, wherein thenanoparticles comprise: about 3 to about 40 weight percent of budesonideand poly(lactic)acid-poly(ethylene)glycol copolymer, wherein thepoly(ethylene)glycol portion of the copolymer has a number averagemolecular weight of about 4 kDa to about 6 kDa, and the poly(lactic)acidportion of the copolymer has a number average molecular weight of about15 kDa to about 20 kDa.